Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

Abstract

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

Description

RELATED APPLICATIONS

[0001] The present application claims priority to provisional application U.S. Serial No. 760/254,553, filed Dec. 12, 2004 (Atty. Docket CL001014-PROV), and application U.S. Ser. No. 09/739,457, filed Dec. 19, 2000 (Atty. Docket CL001014).

FIELD OF THE INVENTION

[0002] The present invention is in the field of transporter proteins that are related to the sugar transporter subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect ligand transport and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0003] Transporters

[0004] Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0005] Transporters are generally classified by structure and the type of mode of action. In addition, transporters are sometimes classified by the molecule type that is transported, for example, sugar transporters, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of molecule (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters: Receptor and transporter nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 (1997) and http://www-biology.ucsd.edu/.about.msaier/- transport/titlepage2.html.

[0006] The following general classification scheme is known in the art and is followed in the present discoveries.

[0007] Channel-type transporters. Transmembrane channel proteins of this class are ubiquitously found in the membranes of all types of organisms from bacteria to higher eukaryotes. Transport systems of this type catalyze facilitated diffusion (by an energy-independent process) by passage through a transmembrane aqueous pore or channel without evidence for a carrier-mediated mechanism. These channel proteins usually consist largely of a-helical spanners, although b-strands may also be present and may even comprise the channel. However, outer membrane porin-type channel proteins are excluded from this class and are instead included in class 9.

[0008] Carrier-type transporters. Transport systems are included in this class if they utilize a carrier-mediated process to catalyze uniport (a single species is transported by facilitated diffusion), antiport (two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy) and/or symport (two or more species are transported together in the same direction in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy).

[0009] Pyrophosphate bond hydrolysis-driven active transporters. Transport systems are included in this class if they hydrolyze pyrophosphate or the terminal pyrophosphate bond in ATP or another nucleoside triphosphate to drive the active uptake and/or extrusion of a solute or solutes. The transport protein may or may not be transiently phosphorylated, but the substrate is not phosphorylated.

[0010] PEP-dependent, phosphoryl transfer-driven group translocators. Transport systems of the bacterial phosphoenolpyruvate:sugar phosphotransferase system are included in this class. The product of the reaction, derived from extracellular sugar, is a cytoplasmic sugar-phosphate.

[0011] Decarboxylation-driven active transporters. Transport systems that drive solute (e.g., ion) uptake or extrusion by decarboxylation of a cytoplasmic substrate are included in this class.

[0012] Oxidoreduction-driven active transporters. Transport systems that drive transport of a solute (e.g., an ion) energized by the flow of electrons from a reduced substrate to an oxidized substrate are included in this class. Light-driven active transporters. Transport systems that utilize light energy to drive transport of a solute (e.g., an ion) are included in this class.

[0013] Mechanically-driven active transporters. Transport systems are included in this class if they drive movement of a cell or organelle by allowing the flow of ions (or other solutes) through the membrane down their electrochemical gradients.

[0014] Outer-membrane porins (of b-structure). These proteins form transmembrane pores or channels that usually allow the energy independent passage of solutes across a membrane. The transmembrane portions of these proteins consist exclusively of b-strands that form a b-barrel. These porin-type proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and eukaryotic plastids.

[0015] Methyltransferase-driven active transporters. A single characterized protein currently falls into this category, the Na+-transporting methyltetrahydromethanopterin:coenzyme M methyltransferase.

[0016] Non-ribosome-synthesized channel-forming peptides or peptide-like molecules. These molecules, usually chains of L- and D-amino acids as well as other small molecular building blocks such as lactate, form oligomeric transmembrane ion channels. Voltage may induce channel formation by promoting assembly of the transmembrane channel. These peptides are often made by bacteria and fungi as agents of biological warfare.

[0017] Non-Proteinaceous Transport Complexes. Ion conducting substances in biological membranes that do not consist of or are not derived from proteins or peptides fall into this category.

[0018] Functionally characterized transporters for which sequence data are lacking. Transporters of particular physiological significance will be included in this category even though a family assignment cannot be made.

[0019] Putative transporters in which no family member is an established transporter. Putative transport protein families are grouped under this number and will either be classified elsewhere when the transport function of a member becomes established, or will be eliminated from the TC classification system if the proposed transport function is disproven. These families include a member or members for which a transport function has been suggested, but evidence for such a function is not yet compelling.

[0020] Auxiliary transport proteins. Proteins that in some way facilitate transport across one or more biological membranes but do not themselves participate directly in transport are included in this class. These proteins always function in conjunction with one or more transport proteins. They may provide a function connected with energy coupling to transport, play a structural role in complex formation or serve a regulatory function.

[0021] Transporters of unknown classification. Transport protein families of unknown classification are grouped under this number and will be classified elsewhere when the transport process and energy coupling mechanism are characterized. These families include at least one member for which a transport function has been established, but either the mode of transport or the energy coupling mechanism is not known.

[0022] Ion Channels

[0023] An important type of transporter is the ion channel. Ion channels regulate many different cell proliferation, differentiation, and signaling processes by regulating the flow of ions into and out of cells. Ion channels are found in the plasma membranes of virtually every cell in eukaryotic organisms. Ion channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, ion channels, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0024] Ion channels are generally classified by structure and the type of mode of action. For example, extracellular ligand gated channels (ELGs) are comprised of five polypeptide subunits, with each subunit having 4 membrane spanning domains, and are activated by the binding of an extracellular ligand to the channel. In addition, channels are sometimes classified by the ion type that is transported, for example, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of ion (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters (1997). Receptor and ion channel nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 and http://www-biology.ucsd.edu/.about.msaier/transport/toc.htm- l.

[0025] There are many types of ion channels based on structure. For example, many ion channels fall within one of the following groups: extracellular ligand-gated channels (ELG), intracellular ligand-gated channels (ILG), inward rectifying channels (INR), intercellular (gap junction) channels, and voltage gated channels (VIC). There are additionally recognized other channel families based on ion-type transported, cellular location and drug sensitivity. Detailed information on each of these, their activity, ligand type, ion type, disease association, drugability, and other information pertinent to the present invention, is well known in the art.

[0026] Extracellular ligand-gated channels, ELGs, are generally comprised of five polypeptide subunits, Unwin, N. (1993), Cell 72: 31-41; Unwin, N. (1995), Nature 373: 3743; Hucho, F., et al., (1996) J. Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem. 239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), Trends Pharmacol. Sci., Elsevier, pp. 4-6; 36-40; 4244; and Xue, H. (1998) J. Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions: this serves as a means of identifying other members of the ELG family of proteins. ELG bind a ligand and in response modulate the flow of ions. Examples of ELG include most members of the neurotransmitter-receptor family of proteins, e.g., GABAI receptors. Other members of this family of ion channels include glycine receptors, ryandyne receptors, and ligand gated calcium channels.

[0027] The Voltage-gated Ion Channel (VIC) Superfamily

[0028] Proteins of the VIC family are ion-selective channel proteins found in a wide range of bacteria, archaea and eukaryotes Hille, B. (1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolution and diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed., Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F. J. (1993), Quart. Rev. Biophys. 27: 140; Salkoff, L. and T. Jegla (1995), Neuron 15: 489492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci., Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. and W. Stuhmer (1998), Naturwissenschaften 85: 437-444. They are often homo- or heterooligomeric structures with several dissimilar subunits (e.g., a1-a2-d-b Ca.sup.2+ channels, ab.sub.1b.sub.2 Na.sup.+ channels or (a).sub.4-b K.sup.+ channels), but the channel and the primary receptor is usually associated with the a (or a1) subunit. Functionally characterized members are specific for K.sup.+, Na.sup.+ or Ca.sup.2+. The K.sup.+ channels usually consist of homotetrameric structures with each a-subunit possessing six transmembrane spanners (TMSs). The al and a subunits of the Ca.sup.2+ and Na.sup.+ channels, respectively, are about four times as large and possess 4 units, each with 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs. These large channel proteins form heterotetra-unit structures equivalent to the homotetrameric structures of most K.sup.+ channels. All four units of the Ca.sup.2+ and Na.sup.+ channels are homologous to the single unit in the homotetrameric K.sup.+ channels. Ion flux via the eukaryotic channels is generally controlled by the transmembrane electrical potential (hence the designation, voltage-sensitive) although some are controlled by ligand or receptor binding.

[0029] Several putative K.sup.+-selective channel proteins of the VIC family have been identified in prokaryotes. The structure of one of them, the KcsA K.sup.+ channel of Streptomyces lividans, has been solved to 3.2 .ANG. resolution. The protein possesses four identical subunits, each with two transmembrane helices, arranged in the shape of an inverted teepee or cone. The cone cradles the "selectivity filter" P domain in its outer end. The narrow selectivity filter is only 12 .ANG. long, whereas the remainder of the channel is wider and lined with hydrophobic residues. A large water-filled cavity and helix dipoles stabilize K.sup.+ in the pore. The selectivity filter has two bound K.sup.+ ions about 7.5 .ANG. apart from each other. Ion conduction is proposed to result from a balance of electrostatic attractive and repulsive forces.

[0030] In eukaryotes, each VIC family channel type has several subtypes based on pharmacological and electrophysiological data. Thus, there are five types of Ca.sup.2+ channels (L, N, P, Q and T). There are at least ten types of K.sup.+ channels, each responding in different ways to different stimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca.sup.2+-sensitive [BK.sub.ca, IK.sub.Ca and SK.sub.ca] and receptor-coupled [K.sub.M and K.sub.Ach]. There are at least six types of Na.sup.+ channels (I, II, III, .mu.1, H1 and PN3). Tetrameric channels from both prokaryotic and eukaryotic organisms are known in which each a-subunit possesses 2 TMSs rather than 6, and these two TMSs are homologous to TMSs 5 and 6 of the six TMS unit found in the voltage-sensitive channel proteins. KcsA of S. lividans is an example of such a 2 TMS channel protein. These channels may include the K.sub.Na (Na.sup.+-activated) and K.sub.Vol (cell volume-sensitive) K.sup.+ channels, as well as distantly related channels such as the Tokl K.sup.+ channel of yeast, the TWIK-1 inward rectifier K.sup.+ channel of the mouse and the TREK-1 K.sup.+ channel of the mouse. Because of insufficient sequence similarity with proteins of the VIC family, inward rectifier K.sup.+ IRK channels (ATP-regulated; G-protein-activated) which possess a P domain and two flanking TMSs are placed in a distinct family. However, substantial sequence similarity in the P region suggests that they are homologous. The b, g and d subunits of VIC family members, when present, frequently play regulatory roles in channel activation/deactivation.

[0031] The Epithelial Na.sup.+ Channel (ENaC) Family

[0032] The ENaC family consists of over twenty-four sequenced proteins (Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H. Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G. Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997), Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273: 9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger, J.-D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are from animals with no recognizable homologues in other eukaryotes or bacteria. The vertebrate ENaC proteins from epithelial cells cluster tightly together on the phylogenetic tree: voltage-insensitive ENaC homologues are also found in the brain. Eleven sequenced C. elegans proteins, including the degenerins, are distantly related to the vertebrate proteins as well as to each other. At least some of these proteins form part of a mechano-transducing complex for touch sensitivity. The homologous Helix aspersa (FMRF-amide)-activated Na.sup.+ channel is the first peptide neurotransmitter-gated ionotropic receptor to be sequenced.

[0033] Protein members of this family all exhibit the same apparent topology, each with N- and C-termini on the inside of the cell, two amphipathic transmembrane spanning segments, and a large extracellular loop. The extracellular domains contain numerous highly conserved cysteine residues. They are proposed to serve a receptor function.

[0034] Mammalian ENaC is important for the maintenance of Na.sup.+ balance and the regulation of blood pressure. Three homologous ENaC subunits, alpha, beta, and gamma, have been shown to assemble to form the highly Na.sup.+-selective channel. The stoichiometry of the three subunits is alpha.sub.2, beta1, gammal in a heterotetrameric architecture.

[0035] The Glutamate-gated Ion Channel (GIC) Family of Neurotransmitter Receptors

[0036] Members of the GIC family are heteropentameric complexes in which each of the 5 subunits is of 800-1000 amino acyl residues in length (Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell 72: 3141; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol. Sci., Elsevier, pp. 36-40). These subunits may span the membrane three or five times as putative a-helices with the N-termini (the glutamate-binding domains) localized extracellularly and the C-termini localized cytoplasmically. They may be distantly related to the ligand-gated ion channels, and if so, they may possess substantial b-structure in their transmembrane regions. However, homology between these two families cannot be established on the basis of sequence comparisons alone. The subunits fall into six subfamilies: a, b, g, d, e and z.

[0037] The GIC channels are divided into three types: (1) a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate- and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Subunits of the AMPA and kainate classes exhibit 35-40% identity with each other while subunits of the NMDA receptors exhibit 22-24% identity with the former subunits. They possess large N-terminal, extracellular glutamate-binding domains that are homologous to the periplasmic glutamine and glutamate receptors of ABC-type uptake permeases of Gram-negative bacteria. All known members of the GIC family are from animals. The different channel (receptor) types exhibit distinct ion selectivities and conductance properties. The NMDA-selective large conductance channels are highly permeable to monovalent cations and Ca.sup.2+. The AMPA- and kainate-selective ion channels are permeable primarily to monovalent cations with only low permeability to Ca.sup.2+.

[0038] The Chloride Channel (ClC) Family

[0039] The ClC family is a large family consisting of dozens of sequenced proteins derived from Gram-negative and Gram-positive bacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer, K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J. Biol. Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol. 242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher, W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998), Annu. Rev. Physiol. 60: 689-717). These proteins are essentially ubiquitous, although they are riot encoded within genomes of Haemophilus influenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequenced proteins vary in size from 395 amino acyl residues (M. jannaschii) to 988 residues (man). Several organisms contain multiple ClC family paralogues. For example, Synechocystis has two paralogues, one of 451 residues in length and the other of 899 residues. Arabidopsis thaliana has at least four sequenced paralogues, (775-792 residues), humans also have at least five paralogues (820-988 residues), and C. elegans also has at least five (810-950 residues). There are nine known members in mammals, and mutations in three of the corresponding genes cause human diseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiae only have one ClC family member each. With the exception of the larger Synechocystis paralogue, all bacterial proteins are small (395-492 residues) while all eukaryotic proteins are larger (687-988 residues). These proteins exhibit 10-12 putative transmembrane a-helical spanners (TMSs) and appear to be present in the membrane as homodimers. While one member of the family, Torpedo ClC-O, has been reported to have two channels, one per subunit, others are believed to have just one.

[0040] All functionally characterized members of the ClC family transport chloride, some in a voltage-regulated process. These channels serve a variety of physiological functions (cell volume regulation; membrane potential stabilization; signal transduction; transepithelial transport, etc.). Different homologues in humans exhibit differing anion selectivities, i.e., ClC4 and ClC5 share a NO.sub.3.sup.->Cl.sup.->- Br.sup.->I.sup.- conductance sequence, while ClC3 has an I.sup.->Cl.sup.- selectivity. The ClC4 and ClC5 channels and others exhibit outward rectifying currents with currents only at voltages more positive than +20 mV.

[0041] Animal Inward Rectifier K.sup.+ Channel (IRK-C) Family

[0042] IRK channels possess the "minimal channel-forming structure" with only a P domain, characteristic of the channel proteins of the VIC family, and two flanking transmembrane spanners (Shuck, M. E., et al., (1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995), Am. J. Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78: 227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171). They may exist in the membrane as homo- or heterooligomers. They have a greater tendency to let K.sup.+ flow into the cell than out. Voltage-dependence may be regulated by external K.sup.+, by internal Mg.sup.2+, by internal ATP and/or by G-proteins. The P domains of IRK channels exhibit limited sequence similarity to those of the VIC family, but this sequence similarity is insufficient to establish homology. Inward rectifiers play a role in setting cellular membrane potentials, and the closing of these channels upon depolarization permits the occurrence of long duration action potentials with a plateau phase. Inward rectifiers lack the intrinsic voltage sensing helices found in VIC family channels. In a few cases, those of Kir1.1a and Kir6.2, for example, direct interaction with a member of the ABC superfamily has been proposed to confer unique functional and regulatory properties to the heteromeric complex, including sensitivity to ATP. The SUR1 sulfonylurea receptor (spQ09428) is the ABC protein that regulates the Kir6.2 channel in response to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 are the cause of familial persistent hyperinsulinemic hypoglycemia in infancy (PHHI), an autosomal recessive disorder characterized by unregulated insulin secretion in the pancreas.

[0043] ATP-gated Cation Channel (ACC) Family

[0044] Members of the ACC family (also called P2X receptors) respond to ATP, a functional neurotransmitter released by exocytosis from many types of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8: 474483; Soto, F., M. Garcia-Guzman and W. Stuhmer (1997), J. Membr. Biol. 160: 91-100). They have been placed into seven groups (P2X.sub.1-P2X.sub.7) based on their pharmacological properties. These channels, which function at neuron-neuron and neuron-smooth muscle junctions, may play roles in the control of blood pressure and pain sensation. They may also function in lymphocyte and platelet physiology. They are found only in animals.

[0045] The proteins of the ACC family are quite similar in sequence (>35% identity), but they possess 380-1000 amino acyl residues per subunit with variability in length localized primarily to the C-terminal domains. They possess two transmembrane spanners, one about 30-50 residues from their N-termini, the other near residues 320-340. The extracellular receptor domains between these two spanners (of about 270 residues) are well conserved with numerous conserved glycyl and cysteyl residues. The hydrophilic C-termini vary in length from 25 to 240 residues. They resemble the topologically similar epithelial Na.sup.+ channel (ENaC) proteins in possessing (a) N- and C-termini localized intracellularly, (b) two putative transmembrane spanners, (c) a large extracellular loop domain, and (d) many conserved extracellular cysteyl residues. ACC family members are, however, not demonstrably homologous with them. ACC channels are probably hetero- or homomultimers and transport small monovalent cations (Me.sup.+). Some also transport Ca.sup.2+; a few also transport small metabolites.

[0046] The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca.sup.2+ Channel (RIR-CaC) Family

[0047] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP3)-sensitive Ca.sup.2+-release channels function in the release of Ca.sup.2+ from intracellular storage sites in animal cells and thereby regulate various Ca.sup.2+-dependent physiological processes (Hasan, G. et al., (1992) Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem. 269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477487; Lee, A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors and Channels (A. G. Lee, ed.), JAI Press, Denver, Colo., pp 291-326; Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ry receptors occur primarily in muscle cell sarcoplasmic reticular (SR) membranes, and IP3 receptors occur primarily in brain cell endoplasmic reticular (ER) membranes where they effect release of Ca.sup.2+ into the cytoplasm upon activation (opening) of the channel.

[0048] The Ry receptors are activated as a result of the activity of dihydropyridine-sensitive Ca.sup.2+ channels. The latter are members of the voltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitive channels are present in the T-tubular systems of muscle tissues.

[0049] Ry receptors are homotetrameric complexes with each subunit exhibiting a molecular size of over 500,000 daltons (about 5,000 amino acyl residues). They possess C-terminal domains with six putative transmembrane a -helical spanners (TMSs). Putative pore-forming sequences occur between the fifth and sixth TMSs as suggested for members of the VIC family. The large N-terminal hydrophilic domains and the small C-terminal hydrophilic domains are localized to the cytoplasm. Low resolution 3-dimensional structural data are available. Mammals possess at least three isoforms that probably arose by gene duplication and divergence before divergence of the mammalian species. Homologues are present in humans and Caenorabditis elegans.

[0050] IP.sub.3 receptors resemble Ry receptors in many respects. (1) They are homotetrameric complexes with each subunit exhibiting a molecular size of over 300,000 daltons (about 2,700 amino acyl residues). (2) They possess C-terminal channel domains that are homologous to those of the Ry receptors. (3) The channel domains possess six putative TMSs and a putative channel lining region between TMSs 5 and 6. (4) Both the large N-terminal domains and the smaller C-terminal tails face the cytoplasm. (5) They possess covalently linked carbohydrate on extracytoplasmic loops of the channel domains. (6) They have three currently recognized isoforms (types 1, 2, and 3) in mammals which are subject to differential regulation and have different tissue distributions.

[0051] IP.sub.3 receptors possess three domains: N-terminal IP.sub.3-binding domains, central coupling or regulatory domains and C-terminal channel domains. Channels are activated by IP.sub.3 binding, and like the Ry receptors, the activities of the IP.sub.3 receptor channels are regulated by phosphorylation of the regulatory domains, catalyzed by various protein kinases. They predominate in the endoplasmic reticular membranes of various cell types in the brain but have also been found in the plasma membranes of some nerve cells derived from a variety of tissues.

[0052] The channel domains of the Ry and IP.sub.3 receptors comprise a coherent family that in spite of apparent structural similarities, do not show appreciable sequence similarity of the proteins of the VIC family. The Ry receptors and the IP.sub.3 receptors cluster separately on the RIR-CaC family tree. They both have homologues in Drosophila. Based on the phylogenetic tree for the family, the family probably evolved in the following sequence: (1) A gene duplication event occurred that gave rise to Ry and IP.sub.3 receptors in invertebrates. (2) Vertebrates evolved from invertebrates. (3) The three isoforms of each receptor arose as a result of two distinct gene duplication events. (4) These isoforms were transmitted to mammals before divergence of the mammalian species.

[0053] The Organellar Chloride Channel (O-ClC) Family

[0054] Proteins of the O-ClC family are voltage-sensitive chloride channels found in intracellular membranes but not the plasma membranes of animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268: 14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272: 12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272: 23880-23886).

[0055] They are found in human nuclear membranes, and the bovine protein targets to the microsomes, but not the plasma membrane, when expressed in Xenopus laevis oocytes. These proteins are thought to function in the regulation of the membrane potential and in transepithelial ion absorption and secretion in the kidney. They possess two putative transmembrane a-helical spanners (TMSs) with cytoplasmic N- and C-termini and a large luminal loop that may be glycosylated. The bovine protein is 437 amino acyl residues in length and has the two putative TMSs at positions 223-239 and 367-385. The human nuclear protein is much smaller (241 residues). A C. elegans homologue is 260 residues long.

[0056] Sugar Transporter

[0057] Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na.sup.+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter, and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome.

[0058] Cotransporters are a major class of membrane proteins--typically with 13 menbrane spanning helices. They cause the concentration of molecules across a membrane--nutrients, neurotransmitters, osmolytes and ions. For example there are co transporters for amino acids, sugars, nucleosides and vitamins.

[0059] Na+/glucose co transporter (SGLT1 ) was reported in 1960 by Bob Crane. Sodium dependent glucose transport occurs in both the kidney and the intestine of animals. Both of these transporters show a close similarity to each other.

[0060] These transporters are reported to be multifunctional and have been shown to operate in 4 ways: 1) Uncoupled passive Na+ transport, 2) Downhill water transport, 3) Na+ and substrate transport, 4) Na+, water and substrate transport. For further information regarding to the present invention, see Matsuo et al., Biochem Biophys Res Commun Sep. 8, 1997 ;238(1):126-9.

[0061] Hexose transport into mammalian cells is catalyzed by members of a small family of 44- to 55-kD membrane proteins that have specific functions and differ in their tissue distribution. Observed hexose transporters have 12 membrane-spanning helices and a number of critical conserved residues. By EST database searching for clones containing conserved GLUT sequences, followed by screening of rat tissues and 5-prime RACE, Ibberson et al. and Doege et al. identified rodent and human cDNAs encoding a novel glucose transporter. (Ibberson, M., et al., J. Biol. Chem. 275: 4607-4612, 2000, PubMed ID: 10671487; and Doege, H., et al., J. Biol. Chem. 275: 16275-16280, 2000, PubMed ID: 10821868) The human cDNA encodes a deduced 477-amino acid protein, designated GLUT8 or GLUTX1, that shares 85% sequence homology with the mouse sequence. Ibberson et al. found that the approximately 37-kD rat Glutx1 expressed in frog oocytes is unable to take up glucose unless the N-terminal dileucine motif, which may serve as an internalization signal, is mutated to alanines. Immunofluorescence analysis demonstrated that Glutx1 is expressed intracellularly, whereas Glutx1(LL-AA) is expressed on the plasma membrane. In apparent contrast, Doege et al. found that membrane preparations from cells expressing GLUT8 cannot bind cytochalasin B in the presence of glucose and, when reconstituted in liposomes, have increased D-glucose transport activity. By Western blot analysis, Doege et al. determined that human GLUT8 is expressed as a 42-kD protein. Northern blot analysis revealed expression of a 2.4-kb transcript, with strongest expression in testis and moderate expression in other tissues except thyroid. In addition, Doege et al. found that GLUT8 was not detectable in 2 patients with testicular carcinoma or in testicular tissue of 4 patients treated with estrogen. They found that Glut8 mRNA was detectable in testis from pubertal and adult, but not prepubertal, rats

[0062] Glucose transport activity in early preimplantation mouse embryos had been attributed to the known facilitative glucose transporters GLUT1 (SLC2A1; 138140), GLUT2 (SLC2A2; 138160), and GLUT3 (SLC2A3; 138170). GLUT1 is present throughout the preimplantation period, which begins with the 1-cell embryo and ends with the blastocyst stage. GLUT2 and GLUT3 are first expressed at a late 8-cell stage and remain present for the rest of the preimplantation period. The simultaneous appearance of all 3 transporters corresponds to the critical time in mammalian development when an embryonic fuel metabolism switches from the oxidation of lactate and pyruvate via the Krebs cycle and oxidative phosphorylation to anaerobic metabolism of glucose via glycolysis. Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulin-regulated transporter, GLUT4 (SLC2A4; 138190). Carayannopoulos et al. found that mouse Glut8 exhibits 20 to 25% amino acid sequence identity with Glutl, Glut3, and Glut4. (Carayannopoulos, M. O., et al., Proc. Nat. Acad. Sci. 97: 7313-7318, 2000, PubMed ID: 10860996) Insulin induced a change in the intracellular localization of this protein, which translated into increased glucose uptake into the blastocyst, a process that was inhibited by antisense oligoprobes. The presence of this transporter may be necessary for successful blastocyst development, fuel metabolism, and subsequent implantation. The existence of an alternative transporter may explain examples in other tissues of insulin-regulated glucose transport in the absence of Glut4

[0063] Doege et al. noted that the International Radiation Hybrid Mapping Consortium localized the GLUT8 gene to chromosome 9 (A005N15).

[0064] Transporter proteins, particularly members of the sugar transporter subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing previously unidentified human transport proteins.

SUMMARY OF THE INVENTION

[0065] The present invention is based in part on the identification of amino acid sequences of human transporter peptides and proteins that are related to the sugar transporter subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate transporter activity in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain.

DESCRIPTION OF THE FIGURE SHEETS

[0066] FIG. 1 provides the nucleotide sequence of cDNA molecules or transcript sequences that encode the transporter proteins of the present invention. In addition structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain.

[0067] FIG. 2 provides the predicted amino acid sequence of the transporter of the present invention. In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

[0068] FIG. 3 provides genomic sequences that span the gene encoding the transporter protein of the present invention. In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs, including insertion/deletion variants ("indels"), were identified at 42 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0069] General Description

[0070] The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a transporter protein or part of a transporter protein and are related to the sugar transporter subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human transporter peptides and proteins that are related to the sugar transporter subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these transporter peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the transporter of the present invention.

[0071] In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known transporter proteins of the sugar transporter subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known sugar transporter family or subfamily of transporter proteins.

[0072] Specific Embodiments

[0073] Peptide Molecules

[0074] The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the transporter family of proteins and are related to the sugar transporter subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the transporter peptides of the present invention, transporter peptides, or peptides/proteins of the present invention.

[0075] The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprising the amino acid sequences of the transporter peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

[0076] As used herein, a peptide is said to be "isolated" or "purified" when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

[0077] In some uses, "substantially free of cellular material" includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

[0078] The language "substantially free of chemical precursors or other chemicals" includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of the transporter peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

[0079] The isolated transporter peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain. For example, a nucleic acid molecule encoding the transporter peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

[0080] Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:3 and SEQ ID NO:4), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) and the genomic sequences provided in FIG. 3 (SEQ ID NO:5). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

[0081] The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:3 and SEQ ID NO:4), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) and the genomic sequences provided in FIG. 3 (SEQ ID NO:5). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

[0082] The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:3 and SEQ ID NO:4), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) and the genomic sequences provided in FIG. 3 (SEQ ID NO:5). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the transporter peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

[0083] The transporter peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a transporter peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the transporter peptide. "Operatively linked" indicates that the transporter peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the transporter peptide.

[0084] In some uses, the fusion protein does not affect the activity of the transporter peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant transporter peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

[0085] A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A transporter peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the transporter peptide.

[0086] As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

[0087] Such variantsican readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the transporter peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

[0088] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0089] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined:using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been. incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0090] The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0091] Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the transporter peptides of the present invention as well as being encoded by the same genetic locus as the transporter peptide provided herein. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 1.

[0092] Allelic variants of a transporter peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by the same genetic locus as the transporter peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 1. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

[0093] FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 42 different nucleotide positions in introns and regions 5' and 3' of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements. Two SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynbnymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

[0094] Paralogs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

[0095] Orthologs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

[0096] Non-naturally occurring variants of the transporter peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the transporter peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute agiven amino acid in a transporter peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, leu, and Ile; interchange of the hydroxyl residues Serand Thr, exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0097] Variant transporter peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to transport ligand, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

[0098] Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0099] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as transporter activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0100] The present invention further provides fragments of the transporter peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

[0101] As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a transporter peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the transporter peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the transporter peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

[0102] Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in transporter peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

[0103] Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0104] Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins--Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth, Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62 (1992)).

[0105] Accordingly, the transporter peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature transporter peptide is fused with another compound, such as a compound to increase the half-life of the transporter peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature transporter peptide, such as a leader or secretory sequence or a sequence for purification of the mature transporter peptide or a pro-protein sequence.

[0106] Protein/Peptide Uses

[0107] The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a transporter-effector protein interaction or transporter-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

[0108] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0109] Substantial chemical and structural homology exists between the sugar transporter protein described herein and sugar transporter expressed in the neonatal mouse hippocampus (see FIG. 1). As discussed in the background, sugar transporter expressed in the neonatal mouse hippocampus are known in the art to be involved in sugar absorption. Accordingly, the sugar transporter protein and the encoding gene, provided by the present invention is useful for treating, preventing, and/or diagnosing neuropsychatric disorders, sigar malabsorption and other disorders associated with hippocampus sugar transporter.

[0110] The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, transporters isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the transporter. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney. A large percentage of pharmaceutical agents are being developed that modulate the activity of transporter proteins, particularly members of the sugar transporter subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain. Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.

[0111] The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to transporters that are related to members of the sugar transporter subfamily. Such assays involve any of the known transporter functions or activities or properties useful for diagnosis and treatment of transporter-related conditions that are specific for the subfamily of transporters that the one of the present invention belongs to, particularly in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney. The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems ((Hodgson, Bio/technology, Sep. 10, 1992, (9);973-80). Cell-based systems can be native, i.e., cells that normally express the transporter, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the transporter protein.

[0112] The polypeptides can be used to identify compounds that modulate transporter activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the transporter. Both the transporters of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the transporter. These compounds can be further screened against a functional transporter to determine the effect of the compound on the transporter activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the transporter to a desired degree.

[0113] Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the transporter protein and a molecule that normally interacts with the transporter protein, e.g. a substrate or a component of the signal pathway that the transporter protein normally interacts (for example, another transporter). Such assays typically include the steps of combining the transporter protein with a candidate compound under conditions that allow the transporter protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the transporter protein and the target, such as any of the associated effects of signal transduction such as changes in membrane potential, protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

[0114] Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab').sub.2, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

[0115] One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant transporters or appropriate fragments containing mutations that affect transporter function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.

[0116] The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) transporter activity. The assays typically involve an assay of events in the signal transduction pathway that indicate transporter activity. Thus, the transport of a ligand, change in cell membrane potential, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the transporter protein dependent signal cascade can be assayed.

[0117] Any of the biological or biochemical functions mediated by the transporter can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the transporter can be assayed. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney.

[0118] Binding andlor activating compounds can also be screened by using chimeric transporter proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a ligand-binding region can be used that interacts with a different ligand then that which is recognized by the native transporter. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the transporter is derived.

[0119] The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the transporter (e.g. binding partners and/or ligands). Thus, a compound is exposed to a transporter polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble transporter polypeptide is also added to the mixture. If the test compound interacts with the soluble transporter polypeptide, it decreases the amount of complex formed or activity from the transporter target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the transporter. Thus, the soluble polypeptide that competes with the target transporter region is designed to contain peptide sequences corresponding to the region of interest.

[0120] To perform cell free drug screening assays, it is sometimes desirable to immobilize either the transporter protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

[0121] Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., .sup.35S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of transporter-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a transporter-binding protein and a candidate compound are incubated in the transporter protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the transporter protein target molecule, or which are reactive with transporter protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0122] Agents that modulate one of the transporters of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

[0123] Modulators of transporter protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the transporter pathway, by treating cells or tissues that express the transporter. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain. These methods of treatment include the steps of administering a modulator of transporter activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

[0124] In yet another aspect of the invention, the transporter proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the transporter and are involved in transporter activity. Such transporter-binding proteins are also likely to be involved in the propagation of signals by the transporter proteins or transporter targets as, for example, downstream elements of a transporter-mediated signaling pathway. Alternatively, such transporter-binding proteins are likely to be transporter inhibitors.

[0125] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a transporter protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a transporter-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the transporter protein.

[0126] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a transporter-modulating agent, an antisense transporter nucleic acid molecule, a transporter-specific antibody, or a transporter-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0127] The transporter proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain. The method involves contacting a biological sample with a compound capable of interacting with the transporter protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0128] One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0129] The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, alteredtryptic peptide digest, altered transporter activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0130] In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (EISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

[0131] The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the transporter protein in which one or more of the transporter functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and transporter activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

[0132] The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain. Accordingly, methods for treatment include the use of the transporter protein or fragments.

[0133] Antibodies

[0134] The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

[0135] As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab').sub.2, and Fv fragments.

[0136] Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0137] In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

[0138] Antibodies are preferably prepared from regions or discrete fragments of the transporter proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or transporter/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

[0139] An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

[0140] Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.

[0141] Antibody Uses

[0142] The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

[0143] Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

[0144] The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

[0145] Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

[0146] The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

[0147] The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the transporter peptide to a binding partner such as a ligand or protein binding partner. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

[0148] The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

[0149] Nucleic Acid Molecules

[0150] The present invention further provides isolated nucleic acid molecules that encode a transporter peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the transporter peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

[0151] As used herein, an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

[0152] Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

[0153] For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0154] Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIGS. 1 or 3 (SEQ ID NO:1 and SEQ ID NO:2, transcript sequences and SEQ ID NO:5, genomic sequence), or any nucleic acid molecule that encodes the proteins provided in FIG. 2, SEQ ID NO:3 and SEQ ID NO:4. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

[0155] The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIGS. 1 or 3 (SEQ ID NO:1 and SEQ ID NO:2, transcript sequences and SEQ ID NO:5, genomic sequence), or any nucleic acid molecule that encodes the proteins provided in FIG. 2, SEQ ID NO:3 and SEQ ID NO:4. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

[0156] The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIGS. 1 or 3 (SEQ ID NO:1 and SEQ ID NO:2, transcript sequences and SEQ ID NO:5, genomic sequence), or any nucleic acid molecule that encodes the proteins provided in FIG. 2, SEQ ID NO:3 and SEQ ID NO:4. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

[0157] In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5' and 3' non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

[0158] The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

[0159] As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the transporter peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

[0160] Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

[0161] The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the transporter proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

[0162] The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5' to the ATG start site in the genomic sequence provided in FIG. 3.

[0163] A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

[0164] A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide. pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

[0165] Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 1.

[0166] FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 42 different nucleotide positions in introns and regions 5' and 3' of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements. Two SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

[0167] As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6.times.sodium chloride/sodium citrate (SSC) at about 45 C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

[0168] Nucleic Acid Molecule Uses

[0169] The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA. and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in Figure, 2. As illustrated in FIG. 3, SNPs, including insertion/deletion variants ("indels"), were identified at 42 different nucleotide positions.

[0170] The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3' noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

[0171] The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

[0172] The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0173] The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

[0174] The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 1.

[0175] The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

[0176] The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

[0177] The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

[0178] The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

[0179] The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

[0180] The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain by a virtual northern blot.

[0181] Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression andlor gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in transporter protein expression relative to normal results.

[0182] In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.

[0183] Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a transporter protein, such as by measuring a level of a transporter-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a transporter gene has been mutated. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney.

[0184] Nucleic acid expression assays are useful for drug screening to identify compounds that modulate transporter nucleic acid expression.

[0185] The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the transporter gene, particularly biological and pathological processes that are mediated by the transporter in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain. The method typically includes assaying the ability of the compound to modulate the expression of the transporter nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired transporter nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the transporter nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

[0186] The assay for transporter nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the transporter protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

[0187] Thus, modulators of transporter gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of transporter mRNA in the presence of the candidate compound is compared to the level of expression of transporter mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[0188] The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate transporter nucleic acid expression in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypemephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

[0189] Alternatively, a modulator for transporter nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the transporter nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain.

[0190] The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the transporter gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[0191] The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in transporter nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in transporter genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the transporter gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the transporter gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a transporter protein.

[0192] Individuals carrying mutations in the transporter gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 42 different nucleotide positions in introns and regions 5' and 3' of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements. Two SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. As indicated by the data presented in FIG. 3, the map position was determined to be on chromosome 1. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos.4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0193] Alternatively, mutations in a transporter gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

[0194] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

[0195] Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant transporter gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0196] Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 ;(1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl, 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0197] The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the transporter gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 42 different nucleotide positions in introns and regions 5' and 3' of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements. Two SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

[0198] Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

[0199] The nucleic acid molecules are thus useful as antisense constructs to control transporter gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of transporter protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into transporter protein.

[0200] Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of transporter nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired transporter nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the transporter protein, such as ligand binding.

[0201] The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in transporter gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired transporter protein to treat the individual.

[0202] The invention also encompasses kits for detecting the presence of a transporter nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the transporter protein of the present invention is expressed in the ovary (adenocarcinoma tissue), uterus (leiomyosarcoma tissue), cervix, kidney cancer tissue (hypernephroma), germinal center B cell, colon, and infant brain by a virtual northern blot. In addition, PCR-based tissue screening panels indicate expression in kidney. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting transporter nucleic acid in a biological sample; means for determining the amount of transporter nucleic acid in the sample; and means for comparing the amount of transporter nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect transporter protein mRNA or DNA.

[0203] Nucleic Acid Arrays

[0204] The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1, 2, and 5).

[0205] As used herein "Arrays" or "Microarrays" refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

[0206] The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5', or 3', sequence, sequential oligonucleotides that cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

[0207] In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5' or at the 3' end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[0208] In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot-blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

[0209] In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

[0210] Using such arrays, the present invention provides methods to identify the expression of the transporter proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the transporter gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 42 different nucleotide positions in introns and regions 5' and 3' of the ORF. Such SNPs in introns and outside the ORF may affect control/regulatory elements. Two SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs). The changes in the amino acid sequence that these SNPs cause is indicated in FIG. 3 and can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference.

[0211] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0212] The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

[0213] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[0214] Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[0215] In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified transporter gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

[0216] Vectors/Host Cells

[0217] The invention also provides vectors containing the nucleic acid molecules described herein. The term "vector" refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0218] A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

[0219] The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

[0220] Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

[0221] The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage .lambda., the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[0222] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0223] In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0224] A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0225] The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

[0226] The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

[0227] The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0228] As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can. increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterotransporter. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:3140 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0229] Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0230] The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0231] The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0232] In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC Kaufman et al., EMBO J. 6:187-195 (1987)).

[0233] The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0234] The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

[0235] The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

[0236] The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0237] Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

[0238] In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

[0239] Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

[0240] While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

[0241] Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as transporters, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

[0242] Where the peptide is not secreted into the medium, which is typically the case with transporters, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

[0243] It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified-methionine in some cases as a result of a host-mediated process.

[0244] Uses of Vectors and Host Cells

[0245] The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a transporter protein or peptide that can be further purified to produce desired amounts of transporter protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

[0246] Host cells are also useful for conducting cell-based assays involving the transporter protein or transporter protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native transporter protein is useful for assaying compounds that stimulate or inhibit transporter protein function.

[0247] Host cells are also useful for identifying transporter protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant transporter protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native transporter protein.

[0248] Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a transporter protein and identifying and evaluating modulators of transporter protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[0249] A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the transporter protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

[0250] Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the transporter protein to particular cells.

[0251] Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

[0252] In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0253] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G.sub.o phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0254] Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, transporter protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo transporter protein function, including ligand interaction, the effect of specific mutant transporter proteins on transporter protein function and ligand interaction, and the effect of chimeric transporter proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more transporter protein functions.

[0255] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

Sequence CWU 1

1

49 1 1473 DNA Homo sapiens 1 atgacccagg ggaagaagaa gaaacgggcc gcgaaccgca gtatcatgct ggccaagaag 60 atcatcatta aggacggagg cacgcctcaa ggaataggtt ctcctagtgt ctatcatgca 120 gttatcgtca tctttttgga gttttttgct tggggactat tgacagcacc caccttggtg 180 gtattacatg aaacctttcc taaacataca tttctgatga acggcttaat tcaaggagta 240 aagggtttgt tgtcattcct tagtgccccg cttattggtg ctctttctga tgtttggggc 300 cgaaaatcct tcttgctgct aacggtgttt ttcacatgtg ccccaattcc tttaatgaag 360 atcagcccat ggtggtactt tgctgttatc tctgtttctg gggtttttgc agtgactttt 420 tctgtggtat ttgcatacgt agcagatata acccaagagc atgaaagaag tatggcttat 480 ggactggttt cagcaacatt tgctgcaagt ttagtcacca gtcctgcaat tggagcttat 540 cttggacgag tatatgggga cagcttggtg gtggtcttag ctacagcaat agctttgcta 600 gatatttgtt ttatccttgt tgctgtgcca gagtcgttgc ctgagaaaat gcggccagca 660 tcctggggag cacccatttc ctgggaacaa gctgaccctt ttgcgtcctt aaaaaaagtc 720 ggccaagatt ccatagtgct gctgatctgc attacagtgt ttctctccta cctaccggag 780 gcaggccaat attccagctt ttttttatac ctcagacaga taatgaaatt ttcaccagaa 840 agtgttgcag cgtttatagc agtccttggc attctttcca ttattgcaca gaccatagtc 900 ttgagtttac ttatgaggtc aattggaaat aagaacacca ttttactggg tctaggattt 960 caaatattac agttggcatg gtatggcttt ggttcagaac cttggatgat gtgggctgct 1020 ggggcagtag cagccatgtc tagcatcacc tttcctgctg tcagtgcact tgtttcacga 1080 actgctgatg ctgatcaaca gggtgtcgtt caaggaatga taacaggaat tcgaggatta 1140 tgcaatggtc tgggaccggc cctctatgga ttcattttct acatattcca tgtggaactt 1200 aaagaactgc caataacagg aacagacttg ggaacaaaca caagccctca gcaccacttt 1260 gaacagaatt ccatcatccc tggccctccc ttcctatttg gagcctgttc agtactgctg 1320 gctctgcttg ttgccttgtt tattccggaa cataccaatt taagcttaag gtccagcagt 1380 tggagaaagc actgtggcag tcacagccat cctcataata cacaagcgcc aggagaggcc 1440 aaagaacctt tactccagga cacaaatgtg tga 1473 2 1377 DNA Homo sapiens 2 atgacccagg ggaagaagaa gaaacgggcc gcgaaccgca gtatcatgct ggccaagaag 60 atcatcatta aggacggagg cacggtatta catgaaacct ttcctaaaca tacatttctg 120 atgaacggct taattcaagg agtaaagggt ttgttgtcat tccttagtgc cccgcttatt 180 ggtgctcttt ctgatgtttg gggccgaaaa tccttcttgc tgctaacggt gtttttcaca 240 tgtgccccaa ttcctttaat gaagatcagc ccatggtggt actttgctgt tatctctgtt 300 tctggggttt ttgcagtgac tttttctgtg gtatttgcat acgtagcaga tataacccaa 360 gagcatgaaa gaagtatggc ttatggactg gtttcagcaa catttgctgc aagtttagtc 420 accagtcctg caattggagc ttatcttgga cgagtatatg gggacagctt ggtggtggtc 480 ttagctacag caatagcttt gctagatatt tgttttatcc ttgttgctgt gccagagtcg 540 ttgcctgaga aaatgcggcc agcatcctgg ggagcaccca tttcctggga acaagctgac 600 ccttttgcgt ccttaaaaaa agtcggccaa gattccatag tgctgctgat ctgcattaca 660 gtgtttctct cctacctacc ggaggcaggc caatattcca gctttttttt atacctcaga 720 cagataatga aattttcacc agaaagtgtt gcagcgttta tagcagtcct tggcattctt 780 tccattattg cacagaccat agtcttgagt ttacttatga ggtcaattgg aaataagaac 840 accattttac tgggtctagg atttcaaata ttacagttgg catggtatgg ctttggttca 900 gaaccttgga tgatgtgggc tgctggggca gtagcagcca tgtctagcat cacctttcct 960 gctgtcagtg cacttgtttc acgaactgct gatgctgatc aacagggtgt cgttcaagga 1020 atgataacag gaattcgagg attatgcaat ggtctgggac cggccctcta tggattcatt 1080 ttctacatat tccatgtgga acttaaagaa ctgccaataa caggaacaga cttgggaaca 1140 aacacaagcc ctcagcacca ctttgaacag aattccatca tccctggccc tcccttccta 1200 tttggagcct gttcagtact gctggctctg cttgttgcct tgtttattcc ggaacatacc 1260 aatttaagct taaggtccag cagttggaga aagcactgtg gcagtcacag ccatcctcat 1320 aatacacaag cgccaggaga ggccaaagaa cctttactcc aggacacaaa tgtgtga 1377 3 490 PRT Homo sapiens 3 Met Thr Gln Gly Lys Lys Lys Lys Arg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys Lys Ile Ile Ile Lys Asp Gly Gly Thr Pro Gln Gly Ile 20 25 30 Gly Ser Pro Ser Val Tyr His Ala Val Ile Val Ile Phe Leu Glu Phe 35 40 45 Phe Ala Trp Gly Leu Leu Thr Ala Pro Thr Leu Val Val Leu His Glu 50 55 60 Thr Phe Pro Lys His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val 65 70 75 80 Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser 85 90 95 Asp Val Trp Gly Arg Lys Ser Phe Leu Leu Leu Thr Val Phe Phe Thr 100 105 110 Cys Ala Pro Ile Pro Leu Met Lys Ile Ser Pro Trp Trp Tyr Phe Ala 115 120 125 Val Ile Ser Val Ser Gly Val Phe Ala Val Thr Phe Ser Val Val Phe 130 135 140 Ala Tyr Val Ala Asp Ile Thr Gln Glu His Glu Arg Ser Met Ala Tyr 145 150 155 160 Gly Leu Val Ser Ala Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala 165 170 175 Ile Gly Ala Tyr Leu Gly Arg Val Tyr Gly Asp Ser Leu Val Val Val 180 185 190 Leu Ala Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala 195 200 205 Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala 210 215 220 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val 225 230 235 240 Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe Leu Ser 245 250 255 Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr Leu Arg 260 265 270 Gln Ile Met Lys Phe Ser Pro Glu Ser Val Ala Ala Phe Ile Ala Val 275 280 285 Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile Val Leu Ser Leu Leu 290 295 300 Met Arg Ser Ile Gly Asn Lys Asn Thr Ile Leu Leu Gly Leu Gly Phe 305 310 315 320 Gln Ile Leu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Glu Pro Trp Met 325 330 335 Met Trp Ala Ala Gly Ala Val Ala Ala Met Ser Ser Ile Thr Phe Pro 340 345 350 Ala Val Ser Ala Leu Val Ser Arg Thr Ala Asp Ala Asp Gln Gln Gly 355 360 365 Val Val Gln Gly Met Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu 370 375 380 Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 385 390 395 400 Lys Glu Leu Pro Ile Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro 405 410 415 Gln His His Phe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu 420 425 430 Phe Gly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile 435 440 445 Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His 450 455 460 Cys Gly Ser His Ser His Pro His Asn Thr Gln Ala Pro Gly Glu Ala 465 470 475 480 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val 485 490 4 458 PRT Homo sapiens 4 Met Thr Gln Gly Lys Lys Lys Lys Arg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys Lys Ile Ile Ile Lys Asp Gly Gly Thr Val Leu His Glu 20 25 30 Thr Phe Pro Lys His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val 35 40 45 Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser 50 55 60 Asp Val Trp Gly Arg Lys Ser Phe Leu Leu Leu Thr Val Phe Phe Thr 65 70 75 80 Cys Ala Pro Ile Pro Leu Met Lys Ile Ser Pro Trp Trp Tyr Phe Ala 85 90 95 Val Ile Ser Val Ser Gly Val Phe Ala Val Thr Phe Ser Val Val Phe 100 105 110 Ala Tyr Val Ala Asp Ile Thr Gln Glu His Glu Arg Ser Met Ala Tyr 115 120 125 Gly Leu Val Ser Ala Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala 130 135 140 Ile Gly Ala Tyr Leu Gly Arg Val Tyr Gly Asp Ser Leu Val Val Val 145 150 155 160 Leu Ala Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala 165 170 175 Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala 180 185 190 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val 195 200 205 Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe Leu Ser 210 215 220 Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr Leu Arg 225 230 235 240 Gln Ile Met Lys Phe Ser Pro Glu Ser Val Ala Ala Phe Ile Ala Val 245 250 255 Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile Val Leu Ser Leu Leu 260 265 270 Met Arg Ser Ile Gly Asn Lys Asn Thr Ile Leu Leu Gly Leu Gly Phe 275 280 285 Gln Ile Leu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Glu Pro Trp Met 290 295 300 Met Trp Ala Ala Gly Ala Val Ala Ala Met Ser Ser Ile Thr Phe Pro 305 310 315 320 Ala Val Ser Ala Leu Val Ser Arg Thr Ala Asp Ala Asp Gln Gln Gly 325 330 335 Val Val Gln Gly Met Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu 340 345 350 Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 355 360 365 Lys Glu Leu Pro Ile Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro 370 375 380 Gln His His Phe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu 385 390 395 400 Phe Gly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile 405 410 415 Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His 420 425 430 Cys Gly Ser His Ser His Pro His Asn Thr Gln Ala Pro Gly Glu Ala 435 440 445 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val 450 455 5 49984 DNA Homo sapiens 5 agtcatactg tattttttac ttgtattttt gttgttttgt gggatttaaa aaatattttt 60 attctgagga tagttgaatc cacaggatac tgagggccag ctgtattcac aacccaaatc 120 acatacaaag cgacaagttc atacacaata ggcctattag aacaggactg ttctctcttg 180 tttatcattg cagcctttct agcacaaagc ctgggacatt ctggacattt agtatgtgtt 240 aaatttctct tactacatta tttccaacag tatttactgc aatctgcaat taccttcctt 300 ttgttttgta actgtgtccc ccactagaat gtaagctctg tgcagatagt gtctcattta 360 ttgatgtatc cctggcatct aataaaacac tgacaacaca agcacccagt aaatattttt 420 tgaatgactg aacaataacc agttcataag gctgataaaa ttggtatagc tagatgaagt 480 atgattttga gggactatga aaatcaaagt aaccacacaa taaattatca gccctctact 540 tccattcaaa acaagctcct gggaattgaa ttatgaaatc tatcatatta ctttctctaa 600 agaacttcaa gttgggtgtc aactaaaaag ttgcaggcga ggcgcggtgg ctcaaacctg 660 taatcccagc actttggtag aactgagtat ctcttgaggc caggtttgaa accagcctgg 720 tcaacataac cagactctgt ctttacaaaa gaaaaattaa aattagccag gcatggtggt 780 gtgcatttgt agtcccagat acttgagacg ctgaggcaga aggatcgttt cggaagaggc 840 tgcaggaggc catgatggca ccactgcact ccagcctggg tgacagagtg agaccctgcc 900 tcagaaaata ataataggcc acgcatggtg gctcacacct gtaatcccag cactttggga 960 ggctggggcg ggaacatcac ctcaggtaag gagttcaagc ctggccaaca cggtgaaatt 1020 ccatctctac taaaaataca aaaaaaatta gccaggcatg gtagtgggga cctgtaatcc 1080 cagctactcg ggaggctgag gcaggagaat cacttgaacc tgggagctgg aagttgcagt 1140 gagccaagtt ggcactattg cactgcagcc tgggcaacaa gagcaaaact ctgtctcaaa 1200 aaataaataa taaaaaaagt ttcaaaatga gaatatatgt ttcaaaacaa gtataatgaa 1260 tatacttatt gattggaaaa tataattaga agtatctatc aggctataaa ttgcttttct 1320 tctcccttcc atggaaatta gttttttttt ccatttttag tcagtatgaa aatacaagga 1380 aaaggaaatt caatcaaatt tactttttaa cattttattt ggaaataatt tcaaacttac 1440 agaaaagttg caaaaacagt acaaagaact catacattca tttactgttt ttccttttac 1500 cctatatatt agttatttat agctgtgtaa caaataaccc caaagcttag tggcttacac 1560 caagtacttt tcatcttaca ctgtttctga gtcaggattc caggagtggc taagctaggt 1620 ggtcctacct ctgggtctct catgaagttg tagtcagcca aaggcttgac caaggttgga 1680 ggatctactt ccaaagtgac tcactccgtg gcatttggta ggaggctaca aacagttcct 1740 ggacaactgg atctctccat aggctgcttg agtgtcctga aaacacggaa gcaggcttcc 1800 ccaggctcca agccccaaaa tgaatgaaaa agagacccgc aaaggaagat gcagtgcctt 1860 ttatgaccta gcctctgaag tcaatactgt cacttctgtt ttgatctatc aagagtcact 1920 aagcctagtc tacactcaag gggaggggaa ttagagtcca cctcttccag ggaggaatat 1980 cattgaatct gtgaacatat cttagaacta ccatacctag tttcagtact tttaaacatt 2040 cgccattttg ctttgtccct ctcttttccc cacctacata tacatacaca tacatgttac 2100 tccctaacca tctgagagta gggagcatgc ggtgtatccc tatccctcgt gtttttctct 2160 taaggaaaag gatattctat tatacaacac ggtagttatc aatatctaat tttaacattg 2220 tgatacttta aagtccactt ccacttgtgt aaattgccct ttctagcaat gttcccatca 2280 aatttatttt taaacaatac agtaaaaacg tagagggcca caaagggtga catcggtcag 2340 gtaaggtatt ttttttggca gggaataaaa aaggtcctgg gtctagggag gtaaacaagc 2400 gtgagccagc tgagttctag cgggggtccc tgaacaccaa aaggacaaga ctgtttctga 2460 aacactacat tatctcttaa gttacccatt acttacggaa aatgattttt tactgttccc 2520 ttcggttcct gtcttggtta gaacacagct ggagattgtg ttaatagctt aggacgtctg 2580 tttccgtgag caggtaacaa ctttttgaaa caaattccct catctgctga agaaggggga 2640 caaaaacggc ccctatcgcc cagaaccgtt gcgaggattt agctagctgg tgacgccgga 2700 gcacgaagtt gtacaggtag ccagcagcac ccacgcgagc ccgcggttac cctggccgcg 2760 cggctactgt agagtgggct ggcggcgagc gggcggggcg gtatcacgcg ggaggggcgg 2820 ggcccgctcg tcggctgatc gcacgattgt gacgcgccgc cggaggcagg ccgggccctc 2880 aagatggcgg cgggcgccca gagcggctcg gcccggcagt agtggtggga cggcactagc 2940 tgctggggcc tgccgccccg ggagtggctg cagcagcgcc aggaatcgag gatggtaaaa 3000 tgacccaggg gaagaagaag aaacgggccg cgaaccgcag tatcatgctg gccaagaaga 3060 tcatcattaa ggacggaggc acggtgagct gagttccgcg ccggcgagcg tccctcgggg 3120 cccccatccg gtctctcctt cagaccccca cactgccgtc tctaggcgtc ccggtgcctc 3180 cctcccttcc cccaccctgt ccgagctgcc ggtgcctcgg ggtcgcggac ccgcatgccg 3240 ccgctccggg aatcgtcctc cgctgctcgg gcttgcggcc tccggggccc gtcctctttc 3300 tttcccgcac ctgccgccct ctgctctggc cgcctctgca ggccctgcgg cctcgaaccc 3360 cacgtgcgcc tccgccgcgg ggaggaatgt gcggggctcc cccggcggcc cgcccgccgc 3420 gcccctcgtc gccgcagcct cgcctcgcct tcgccgccag gccccgcgga gccgtcgccg 3480 cgcttgtcaa ggggctggga accatccctg ctctcccata tgttgctaac ggggtggcgt 3540 ctggcgcggg gatcccgctg cggccccgta gtacgttcgc tttctgtttc cacgtctctc 3600 tgcgtcggtg ctccggctct gggctgctta cagtaaaccc tgaccggaga tgggcttccc 3660 tcacttcccg gagtcggaag catgacggca gacacctggg gcctacattc gaacctgcta 3720 gttttcaaag aaaagtcatc actgtgtgtc ttaagatcaa aagtattaga atcagtcatg 3780 gcctaaggat cggaggagga cactttgaag ggaagaaagg tttgcttttt agaaacagtt 3840 gtcatcacag taaactttat gcagtgtgta gttaaccagc tggggacgta ggatttttaa 3900 ttgaaaaaca aaacaaaaca aaactgtttt atgctaacat ttctccgttg ctacactgtg 3960 tggtctttgt tgcatccgct gataccgcgt tctgaaatag aatggaaagg tgatatatat 4020 gtttcactta cctgaagtgt gcagaaattg taccattaat tccatttctg tttatatctt 4080 attggagccg cgatcaactg ctagcacagt agtaaatgtg taagtaggcc accattgagg 4140 atttgctgaa ttcagttgaa aaacgtgaca aaattttatg acatttcaga acacggccca 4200 gtcaatatgc caaagtttag aaaacttgag acatatgtaa tgactttgga atatattttt 4260 agtttaacgt ttattatatg ttatagcttt gacatttatt gaaaaaaaga aacaaattcc 4320 tcaagttctt tttattgaac ttgattaatt aaaacattac tttgattaga tcggttatga 4380 agagtcatag ctcttttgac caagtaggta agaactatgt ggggagaaaa atactgttgc 4440 ctttgtctac ctttagaaag agacaatatt ttacattctt cataaaatct acaaaatagt 4500 ggcaatgaaa gattgtattt tgtaagacca agtgatattt aagatcagta ttttttacaa 4560 aatgtaagaa tgaaactgat taagaaacac agcttccttt tccttggaaa gttcagtttt 4620 attacctttc tttggggttt tgtttgattt gctttacagc agatgctttc tttccaaatc 4680 ctgtgagttt tggaaaagat cgtttttaaa ctttcttgtc ctattattaa ggttgtaatt 4740 aattcttagc ctgctttggg acacaaaata aaatgtttgc accagcaata ggtttcacat 4800 agaacaaatg aagacttttc ttgagggctg tgaacatggg ggctattatc atttctcatc 4860 tttatacact taatatttca ttctctattc taagagcact gggcactcct ttagaaaagg 4920 ggctttgttt tgtatgtttg gatcccacag ggcctagtat gtgaatttta aagtgataaa 4980 aacacttcta ttttgtacta gcacattcct agatgaattt ttattgtaat tttgtttatt 5040 cttatacgta atcagaggat atatttcaat aaatatcagg ggaatatttt gcattatttg 5100 tattttaatc catcccagct ttaaatttaa aaagtataac tattgcagtc atagaaatga 5160 ttgtaaaatg gtagttgctt atctacctct ctacttacaa tagttcagac tactattatg 5220 aacttttttt gtttgtttgt ttgagatgga gtctcactct gttgcccagg ctggaggagt 5280 gcagtggcag gatctcggct cactgtaacc accgcctcct gggttcaagt gattctcctg 5340 cctcagcctc ccgagtagct gggactacag gcacgtgcca ccatgcctgg ctaatttttt 5400 atattttcag tagagacaaa gtttcaccat attggtcagg ctggtcttga actcctgacc 5460 tcatgattca cccaccttgg cctcccaaag tgcagggatt acaggtgtga gccaccgtgc 5520 ccagactgaa cattttttaa gaaaggggaa aaaattgcca tttgatactc tgttgttgtg 5580 tgttttttaa ttcatcgtat catagaatat ttcagtgcta ttgctgttga cctcagagtt 5640 tcagagtttt tataaagttc cgccaatggg tagattcatt cagtgagatg tctgaggctc 5700 tatggtcggt acatgacagt cgtgaacagt atttcacata cctggtcaat ggtactgatt 5760 tgatccccct tctgatttct tcttttcaac aatgttaata aaattctttc ccgttgtcct 5820 gctaatgaca tatatgtaag cctatttggc cagtttaaat atttataaac aaaactagta 5880 agagttgtta atgatttttc tgaaaattag agcagattag agcagatttg tagttttcaa 5940 cggctgaaga aataaatcct tctaaatgag ccagattaat cgtaagttac tgattttttt 6000 attgaaattg tatttcattg aattgtattt cattcagctg aatgaaaaac aggccaggat 6060 aaagctaaca agtaggctac ctatgtgagt agacacaatt aagataaatt acattaaggt 6120 gtgtgatttt atattaggtg tttttaacct gggtctgttc accctgaagt tgtttgcaaa 6180 attttctttt ggctatacat gtttcttgga agagtcccaa aaggtccata cttcccaaaa 6240

gtttaagagc aattgttctg tttgaaaaca gcataagtaa ctaaagaata agttccacat 6300 attatattca gtaaatattt aatcatatac tgtatactac ttcactgatg aaagtaacca 6360 tattagtgaa tttgctttta aagcatccat atatagaaat agtttttagg ccaggggcag 6420 tggctcacgc ctgtaatccc agcactttgg gaggccagtg tgggcagatc acttgaggcc 6480 aggagtctga gactagcctg gccaacatgg tgaaacccca tctttaccaa aattacaaaa 6540 atgagctagg tgtggtggta tgtgcctgta atcccagcta cccgggaggc cgaggcacga 6600 gaatcacttg aacctgggag gccaagattg cagtgagcct agatcacgcc actgcattcc 6660 agcctgggtg atggagtgaa actgtctcaa aaaaaaaaaa aaagaagttt ttagttacag 6720 gttttcatgt atgtaacatt cagtgtaggt atttaagaca gctgaaataa aaataccttc 6780 tgacattttc aaatactaga attctgtttt gttttattaa agcattacca cttgttttta 6840 agcattcctg ttagaggcta agagctaaag agttatttac agtattcaaa ttgaattttc 6900 cttatctttt aaaatgctca tcttaaaata tgatctttat tgttttggcc atacaattgt 6960 ggaactacat ctctgacagt ggaaaatgta tagttctttc agaagtttgt ggtaaaatga 7020 ctttaaagat ttgatagaaa gtaaggcata tctgaattgc atggtcggaa gtacctgaaa 7080 aaagtaaaat tgatatatca tttgaaaatg aaatgcatat ccctggataa gcagagcacc 7140 agattttttt tttcttggca tccctgattt taattaaata ggagtcagca accgtttcaa 7200 gagcaggacc caagctctga ccctttgcac tcttcacctg caaggatggc tgaagtagtg 7260 gcaggaaagc tctctgggat gtagggcctt tgtagaccca gagagctgtt aaataacctt 7320 tggttgctag catgcaagca ataagaaggg cctgtggtgc ttttcttttt ctttcttttt 7380 ttttttcttt tgagacagag ttttgctctt gttgctcagg ctggggtgca atggcgtgat 7440 cttggctcac agcaacctct gcctccctgg ttcaaggaat tctcctacct tagcctcctg 7500 aatagctggg attacaggca tgtgccatca tgcccagcta atttttgtat ttttttagta 7560 gagaccggat tttaccatgt tggccaggct ggtctcgaac tcttgacttc gggtgatcca 7620 cctgcctcag tcttccaaag tgggattaca ggtgtcagcc actgcgcctg gccccgtggt 7680 gcttttcaaa aagcctagaa acatcagggt gtttatattg tctttggcag gtgtgtggct 7740 ggcagcatca ttaattactt agctccttac ctccatggtt cagtgtttgg tttagattgg 7800 tgtgtttggg gataaattaa tatgcagttt ttttttcaga tggctatatg catccagttc 7860 atcctcatgt agttagaaga cttgcatacc aacataatca gaccgtctgc agaaattctc 7920 ctacagttga aatgtaactc ctttgcagct actgaaagtt taaagtttaa gtaaaaaaat 7980 gaatagcttt cttcaggtaa cattctgaca agtctgtatg atttaaaagt ttcaattata 8040 aggaactctg attgtctttt agcattattt taaattggaa gtgtgaaagt aacagttgac 8100 agtttcagcc agggtacatc aagaagagat gaatatgggt ataatatagc tctcaaaatt 8160 tccagtactt tataacaaag aaatatccct cccactgccc tgttttttaa aaaataaata 8220 atacatgttt tccttccagt cgtgggaaac ttaatagaat ggttcaggag ggacaagtat 8280 atgcagcata cctgtcattt tccattcaag ttttacttta tttttaaaat ttattatttt 8340 ttaaaatatt tcaatagttt tggggtacag gtgggttttt ggttacatag atgtttttta 8400 gtgatgattt ctgagatttt agtgcacctg tcacctgacc agtgtatact gtacccaata 8460 tatagtcttt tatccctctc aagcttcccc cccatcctca aagtccattc tattagtctt 8520 acgcctttgc gtcctcatag ctgaactctc acttgtaagt gagaacatac gacatttggt 8580 tttccattcc tgaattactc acttagaata atggcctcca attccatcca agtttctgca 8640 aaagacatta tttcattcct ttttatggct aagtattcaa tggtatatat acaccacatt 8700 ttctttatcc acttgttggt cattgggcac ttgggttggt tccatatctt tgcagttgtg 8760 aattgtgctg ctataaacat gcatgtacat gtgtcttttt catataatga cttcttttac 8820 tttgggtggg tacccagtag tgggattgct ggatcaaaca gtagttctat ttttagttct 8880 ttaaggaatc gccatactgt tttccatagt ggttgtacta gtttacattc ccaacagcag 8940 tgtcaaagtg ttcatttgtc accacatcca caccatctat tattttttga tttttaaatt 9000 atggccattc ttgcaggagt aaatgatatc tcattgtggt tttaatttgc atttccctga 9060 taattggtga tgttgagcat cttttcatat gtttgttggc ttattgtatg ccttttgaaa 9120 aatgtctatt catgtctttt gcctactttt gatgggattg tttgtttttt ttcttgctga 9180 tttgagttcc ttgtagattc tgggtactag tcctttgtca gatgcacagt tcataaatat 9240 tttctccaac tgtatgggtt gtctgtttac tctgctgatt tttttttttt tttttttttg 9300 agatggaatt ttgctcttgt ttcccaggct ggagtgcaat ggcatgatct tggctccctg 9360 caacctctgc ctctcaggtt caagccattc tcctgcctca gcctcccaag tagctgggat 9420 tacaggcaca caccaccatg cctggctaac ttttttgtat ttttagtaga gacgagtttt 9480 ctctatgttg gccaggctgg actcaaacta ctgaccttag gtgatccacc cgccttggcc 9540 tcccaagatg ctgggattac aggcatgcct aggcggctat aagtattttg ctttatttct 9600 gggttatctg ttgtgttcca ttggtcttca tgcctatttt tataccagta ccatgcggtt 9660 ttggtaactg tagccttttg tataatttaa agtcgggtaa tgtgatgcct ccagatttgt 9720 tttttgctta gtcttgcttt ggctatgtgg gctctttttt ggttccatat gaattttagg 9780 attgtttttt cttgttctgt gaagtatgat gctggtattt tgatgggaat tgcattgaat 9840 ctatagattg ttttggtcag tatagtcatt ttcacaatgt tgattcttcc cttccatgaa 9900 catgggatgt gtttcccttt gtgtcattta tgatttcttt taacagtgtt ttgtactttt 9960 ccttgtaaag atctttcact tccttggtta agtgtattcc taggtgtttt gttttttttg 10020 cagctattgt aaaagggatt gagttcttga tttgattctc agctttgtcg ttgctggaat 10080 atagcagtgc tattgatttg tgtcattgat tttgtatcct gagactttac tgaatcgttt 10140 atcagatctc ggagcttttt ggatgcgtca ttagggtttt ctaggtatac agtcatatca 10200 ttggcaaaca gtggcagttt gatttcctct tttccaattt gcatgctcgt tattcctttc 10260 tcttgtctga ttactctggt taggacttct aaatttttta attactatgg gtacaaagta 10320 gatacagata tttatcaggt acatctgata ttttgataca agcatatgtt gatacaggta 10380 tacagtgtat aataaatcag ggatactggg gtatccatta cctcaaactt ttatcatttc 10440 tttgtgttag gaacatgcca attccacttt tattttattt tattttttat tttttgagac 10500 agagtctcgc tctgtcgccc aggcgacata catagtacag tagtgtactc cagcctgggt 10560 gacggggaga ctctgtctca aaataaataa ataaataaat aaataaatct gttcagacta 10620 atgtcctaga gtgtattccc aatgttttct tctagtcgtt tgtggtttca ggttttagat 10680 ttaagtcttt aatccatttt gatttgattg ttgtacatgg caagaggtag gggtataatt 10740 ttattcttct gtatatggat atccactttt cctagcacca tttaggagac tatccttttc 10800 ccaatgtata cttcggtgcc gttgtcaaaa atgagttgac tgtaaatgca tggatttatt 10860 tctgggttct ctattgtgct ctattgtcta tgtatctgtt tttataccag tattatgctg 10920 ttttggttac tatcactttg tagtataatt tgaagtgaag taatgtgatt cctccaagcc 10980 tcggtttttt tttttttttt tttttttttt tttttatgag acagagtcta gctctgtcgc 11040 ctaggctgga gtgcagtggc gcaatcttgg ctcactgcaa cctctgcctc cctggttcaa 11100 gtgattctcc tgcctcagtt tcccgaggaa ctgggattac aggtcccacc accacgcctg 11160 gctaattttt gtatttttag tagagacggg gtttcacttt gatggccagg ctggtcttga 11220 actcctgacc tcaggtgatc cgcccgcctt ggcctcccaa agtgctggga ttacaggcgt 11280 gagtcactgt gcccagcctc cagccttgtt ctttttgctc aggattgctt tggctgttct 11340 ggctcttgtg gttccatata agttttagga tttaaaagaa aaaattctgt gaggaatgtc 11400 atttgtagtt tgatagtaac tgcattgaat ctgtagattg cttttggtag tattaaaatt 11460 ttaacagtat tgattcttcc aatttatgaa catgaaatat cttcccattt gtgtgtgtgt 11520 cctcttcaat tcgtgtcatc aatgttttgt agtctgtaga catctttcac ttctttaagt 11580 ttattcttag gtattacatc tgtagctatt gtaagtggga ttattttctt ggtttctttt 11640 tcagatattt gctgttggca tatagaaatg gtactgattt ttgtatcctg caacttcagt 11700 gaatttgctt ccattctgat agttttttgg tggagtattt agggttctct ctatataagg 11760 tcatgtcatc tgtaaagagg gacagttttg acttcctgtt ttctaatttg catgcctttt 11820 atttcttact catgcttaat tgctctagtt ggtactttcc agtactttgt tgaataagag 11880 tggcgaaagt gggcatcttt gtcttgttcc agatctttga ggaaaggctt tcaggttttc 11940 cctgttcagc atgatagctc tgtgtctgtc atatatggct tttatcatat tgaggtatgt 12000 tccttctata ccatttttga gagtttttat gaagcagtgt tgaattttag taaatgcttt 12060 ttcatcatta attgaaatga tcattttatt ttccttcatt cttttgaaat gatgtatcac 12120 cttgatagat ttatgtatgt tggactatcc tttcatacct ggatgaatcc cacttgaaca 12180 tgatgaatga tttttttgtt tttaattttt ttgagacgga gttttgctct tgttgcccag 12240 gctggaatgc aatggcgcaa tcttggctca ccgcaacttc cgcctcccgc gttcaagcga 12300 ttctcctgcc tcagcttcct gagtagctgg gattacaggc atgcgccacc acgcctggct 12360 aattttgtat ttttagtgga gacggggttt cttcatgttg gtcaggctgg tcttgaactc 12420 ctgacctcag gtgatccacc cgctttggcc tcccaaagtg ctggaattac aggtgagagc 12480 cactgcgccc ggccgatgaa tgatcttttt taagacctcc ttcctgaagg aggtttgcta 12540 gtattttgtt gaggattttt gcatcaatgt tcatcagaga tattgtccca tagtttattt 12600 tgtttttctc catgctagtt ttaggtaatt tttctcttaa ataaacaaag cattttcctc 12660 ctaaagtgca agcatgctta ttagaaaaga tatggaaaat tcagaatagc atagtaaaca 12720 atgtgatatc acttaaaatc attacctaat ataaatttta tttacattga ggtcagtatt 12780 tattgttttt cagagttgaa attaccctac ctatacatgt tatatcctac tttgattttt 12840 aaaaaaatta gcatgcttta agccctgaga agttgtacca agctttctgc taggggctgg 12900 gtatatgtgg tggtgaacat ggtggacaaa acaggtttaa agcttatcaa atttgtggcc 12960 aatttttttt tttttttcag agtctctgtc gcccaggctg gggtgcagtg gtgggatctc 13020 aactcactgc aacctctgcc tcccaggttc aagcgattct cctgcctcag cctttctgag 13080 tagctgggat tacaggcaca cgccaccatg cccagctaat ttttgtattt ttagtagaga 13140 cagggttttg ccatgttggc aaggctggtc tcgaactctt gacctcaagt gatctgccca 13200 ccttggcctc ccaaagtgct gagattacag gcatgagcca ccatgcctgg cctcctgtgg 13260 tctttttttg accttatatt actatgcttg ttccatttga tactaggcat cacctcctcc 13320 ttcctgaaac tgctccattg tcatatgtga cactgtattc tcttcactct cctgatattt 13380 tcttactgtt ccttttatct tcctcttttc tttatcaaag aagaaaaacc ttcactattt 13440 tcctgtgcct cctacttaaa atgttggcgc ttcttggggt tctgtctcag cccactgctg 13500 ttttcacact tgacactcct gataatctca tctactctgg tggtttcaga tatcacattt 13560 gctgctaatt gttttaatca gtgctcaaag acaatacaaa tgtttcaagt aaagagggca 13620 gttttgtaga taggacctga agtaaatctg agcctcgtgg ggggaagtgc tgggaagcca 13680 ccagctttaa ctgctagaca accaagctaa acacttggaa gttgttcttg attctccctt 13740 ccgctattta tcaagctcct cccaatttca aatcctgaat ccttaatccg ttccctcccc 13800 tccaacattc atactgtgcc actgttttat gccctcattt cttgtttgag ctgattcaga 13860 tagcttcctt ttagatgcgc tttgcttctc cattttatcc tttaggaaat caccagagtg 13920 ataatactgc agtgagtctt aagacatctc tggcagcggt ataaacttaa ttttgtattt 13980 tctttctcat gtatatcaaa ttccaaatct cttacatact ttcgctgggg attgttctgc 14040 ttttgagcca tgttgatatc gtgtttatat ttttgccact tgcttcattt atggtttttt 14100 tttttttttt tggttacatc tttgccagaa taatcttaaa actttcatct gattgtgtca 14160 gtcttaatat cttttagtgg ctccccatgg ccttcagaat taaatataga ctccttagca 14220 tggaagctgg tctttgagta cctgtagctt gtctttcaat acacccaacg tgcagcccat 14280 gcactggttg tactgaactc gatatatgag acccataatg ccgcaagcct tggaagcttt 14340 gtacaggctg agccatcttt tccaccctat acctccgcct gtctaactct gttgtgtcct 14400 ttcagccttc ctcctggaag tctgatattt cccacctccc aagctccctt ggactctgta 14460 tgttccaact gcatactgtg cttatgctaa tgaatttcgt tgttgccttg tctgtccctc 14520 tgactttgaa gacagaggca gtgagtacag atgtttgaca cagtgcccag tacatatatg 14580 atcttaatat ttgttgacta ttaacatcgt tgttattgtt aataattata gaatgtactg 14640 ttaacttttt ttaacttttt aaaaaatctt gttttttata gcctcaagga ataggttctc 14700 ctagtgtcta tcatgcagtt atcgtcatct ttttggagtt ttttgcttgg ggactattga 14760 cagcacccac cttggtggta agtaatcttt taaattattt aacactgact ccaaaatctc 14820 ttcttcttca gttttggagg aaaatgtggg ccttttccct ttgcacggtt aattctccca 14880 ccagtattgt tcagtattca ccagtatttt actggttgtc ttttccaact gttaactctc 14940 ccttaccttt ttttgggagg ggggtggcgt ggaggtgttt gaatttggac ttgtcactgg 15000 gcatgttcaa gcagaggctc tgtaactact ctgagtaaaa tggaagagat tcttaaaccg 15060 acaggtttag aaaagatgat gtctgtgacc tgcatgactc ggcataatta ctttgaggtt 15120 catttatgca gctgtacttt ccaaaaacag gtttctgttc atttgggcta agtacctaga 15180 agggctattc tttaatagat ctaagctgat tttacccaaa ttctcccagg tttgaaactt 15240 tagaaaagac ctccctgccc gaccaaacaa ctcagaagat agccagtttt cttatattgg 15300 tgtagataag gggaatggaa ggagggaagg actatctatg gtaaatatct ataccatctt 15360 gaaaggagta attatgataa atgtacagtt taccaaatcc tagaggaata gagttttaaa 15420 gtaatatact atgttttcat gaaggttttt ataaaaaagt tatttaatag aaaaattatg 15480 taagtagatt gaactagcct aagaacattt acagtacata tttcttgata tatttattga 15540 cagctgtgta attgttacta tctatacata aaatattgat gtttagcagt tgcttatgcc 15600 tgtaatccca gcattttggg aggctgggtg ggcagatcgc ttgagctctg gagttgagac 15660 cagcctgggc aacatggtaa aaccttgtct ctacaaaaaa tgcaaaaatt agttgtgcat 15720 ggtggcatat gtttgtagtc ccagctactc gggaggctaa ggcaggagaa tcacttgagc 15780 ccaggaggca gaggttgtag tgacccgata tcgtgccacc acactccagc ctgggcgacg 15840 ggagtgaaac cttgtctcaa aaaaaaaaaa caaaaaaaaa aacagccggg cgcggtggct 15900 cacacctgta atcccagtac tttgggaggc caaggcgggt ggatcacgag gtcaagagat 15960 tgagaccatc ctgaccaaca tggtgaaacc ctgtctctac taaaaataca aaaattagct 16020 gtgcgtggtg gtacgcacct gtaatcccag ctacttggga ggctgaggca ggagaatctc 16080 ttgaacccgg gaagtggagg ttgcagtgag ccgagactgc accactaccc tccagcctgg 16140 atacagggtg agactctgtc tcaaaaataa aaagtcattt tgaatatata gagcatgttc 16200 atgagtattg ctataaaaaa atatcagagg gttttttttt tttttttagt ttactgattt 16260 cagatagaaa tctttaaaaa attaatttac acatttcctg gcttcataat ccaagtacaa 16320 cgatttggaa cttcctcaga tgatgcaagt tgattatgac attcataact tcattgaatt 16380 gtaataacct gtttttgtca agggttactg aagtgctgta ataacttttt gggctcatga 16440 ctttacatta gctttcctaa tgcgccagcg tgctttttat aatctgtcag tttaacatac 16500 aaatctgtct ggtagaccat cactcctacc atttaaagta cttgagcttt gtaatagtaa 16560 acagcccacg tgtatttata ttatgatgga tctaggcaca gtcctttaat gtattcaaag 16620 tggactatgt taagcacagt cctaatgtat tccactttga atacattatc attttttcat 16680 ccttacaacc accttatcag tgagatatta gcctcataat acagatgagg aaaccggggc 16740 ttagaaaagt taagcaattt gattgctact ctgacagtaa gctgcagtgt tggtatttgc 16800 acccaggctt ccttgactcc tccagtgctc agtcttttgg ggaatgcagg tagtaacttg 16860 tttgtaccca tgttttagat agttgaggtt gtcaggcagc ccaaccacta gctaagtagg 16920 gtgatcaaaa tgtggatgag ctgttagcaa gctatgaaaa aaagcatttt gtgatgtttc 16980 cataatttgt tatcagtatt tcaagtgtgt atagctattt ttaaaatttg cttcttgttt 17040 aaattttttt aggtatgtta tctttcgtgt tattttggta catttttttc ctagttggac 17100 aaagggaggc tatctttttt aagaacaagg aaggagtccc cttaattaga aaggcttgtt 17160 tattcatttt tcatagacta atgtgcttaa tatattcctt tttttttttt tttttttttt 17220 tgagacggag tctcgctctg tctgtcccca ggctggagtg cagaggcacg atcttggctc 17280 actgcatccc ccacctccca ggttcaagtg attttcctgc ctcagcctcc caagtagctg 17340 ggactacagg cacatgccac catgcccagc taatttttgt acttttagta gagatggggt 17400 ttcaccatgt tgaccagaat ggtctcgatc tcttaacctc gtgatccgcc cgccttggcc 17460 tcccaaagtg ctgggattac aggtgtgagc cactgtgcct ggccaatata ttcttattat 17520 ctttaatttt tgttttcttt ttcttttttt ttttattttg tttgtttgtg tattttgaga 17580 tggagtctca ctctgttgcc caggctggag tgcagtggtg caaccttggc tcactgcaac 17640 ctccacctcc caggttcaag caattctcct gcctcagcct cccgagtagc tgggactata 17700 ggcacgtgcc aacataccag gctaattttt gtatttttaa tagagacggg gtttcaccac 17760 attggccagg ctggtcttga actcctgacc tcaggtgatc cgcctgcctc ggcctcccag 17820 agtgttggga ttacaggcat aagccaccat gcccagcctg gcatatctac tttttagaag 17880 tgaccctgtt atatattcag tatatgtcac taattaagaa caatatatta attcaatatg 17940 ggctttttaa aaaggtttta ctcatttcaa ggctttttgc ttacaaattt tgtttttttg 18000 ttgttggctt tgttggcagt ctttgttttg ggccccagta ctcctcccac tcctccccag 18060 cattgtgtgt gagaggtgtg taaagaggtg ggtttctggg taaaagaagg ctctcctctt 18120 aaaagtctgt taatctttaa acatttcatt tctgttttat gtgttttgaa actgattata 18180 aatggtgcat gccacaagag tcaaagtttt taacattcat tttaaaagga aaatgaggat 18240 gaagacataa tttaatttat attttaagtc agtatctttc atttccctgt ccctccctca 18300 acagttatat catagtttgt ttcagcattt cagatttcaa agatattctt tgaagtattt 18360 ttttaatcag ataaccagtt ttagacatat taattttgaa tgtctggttt gggatttatg 18420 atagccttaa tttcttaatt tttaaaacta atgtgacatt ttaagaccaa aaaaactgtg 18480 tgttgcaatt atctttcact tttaagccct catagaacag tcaaaaaaca aaagctgtgt 18540 tttgtggaag atctgcccag gggaagatgg tgagcctcta ccaacaaggg gatttagcta 18600 aaaagaagga ttttgtactg acaaatattt ttaaagattg aggtctaaca cttttgagag 18660 gttatgaata tatggttggt catagtagat agttcagtca gaatcagtga ttattgcttg 18720 attatgtaac atattagcta agtgatgaga ataacagtag gtataaggat ctgtaatgcc 18780 aaggagtgga atttaccggt tttttttttt ctttcctttt tttttttttt cattgagacg 18840 gagtcttaat ctggcatcca ggttggagtg cagtggcgtg atctcggctc actgcaacct 18900 ccaccgccaa ggttcaagag attctcctgc ctcagcctcc ccagtagctg gaattacagg 18960 tgcatgtcac cacgcccagc taattttttt ttttattatt ttttttgaga cagagtttca 19020 ctctgtcgtc taggctggaa ttcagtggca ctatctcggc tcactgcaac cttcgcctcc 19080 caggttcaag cagttctctg cctcagcctc ccaagtatgt gggattacag gcacctgcca 19140 ccatgcctgg ctaatttttt tatttctagt agaggcgggg ttttaccatc ttggtcaggc 19200 tggtcttgaa ctccttacct tgtgatccac ccacctaagc ctcccaaagt gctgggatta 19260 caggcgtgag ccactgttcc tggccggctt tacccttttg acagacctat ggctctggaa 19320 ataataggcc agtgtttgat ggttcaagct cctagataca cagtccatgt tacggaacac 19380 tcaaaatcca ctagcatctc ttctacctag atggtttcgt gtccttggct acagaaacag 19440 ccccaaagcg tttaacattt taaggattat ttactttcaa catttttaaa gttaaaaaaa 19500 agttaagatc cataaaattt tttggaaaag tgttacattt tctctgttca cctctaaaga 19560 ccagtgctaa aggatcctga catcaaaaat ctttacaaca ttcgaattac ttgttatatt 19620 tgtctgttaa aattttgtta gaaattgtat ggccccaaag gagaaattgc tttggagaaa 19680 aaagttaggt agcagaggaa cagtttggaa gggttggggg ttggccagat aaagaaaggg 19740 aagaaacatt caaaattgaa aggatgccgt gtataaaata tgaatattgg aaagcataga 19800 atatttcaga aacagtgaag cgaacagatt gattggaatg gaatacagct tggcaaagtg 19860 aatcattagt gataagatct agcatagtat aaaacttctt atagacattc ataatgtttt 19920 tcattctttc taacaccaaa cctgttcttc atacctagaa agatttggct tgcagtaggc 19980 cctatgtgat tattgaaaga aaagcataat acatttgagt cccgtaaaaa gttttgagat 20040 actagtttaa ggagtttaaa tcttatcctt tagcacaagg actgggaaaa tatggctaga 20100 ggactagatc ttttttgcaa tttttttttt ttttttgtag ttgcttctgg caactttctc 20160 tttgtgtgtg tgtttatatt ccttttcaca agtatgttga attgaacttt ttcctaatta 20220 tcacttagct acttagttaa tgcatgcagt agaactctaa aaagaacttc taggagtttc 20280 tcaaagacct cccagtaatt cttttcaatt agagagggca tgccattttt ccttttttat 20340 ttttaaataa tattttattt tttatttttg tgggtacata ggtgtatata tttatggggg 20400 tacatgagat attttgatac aggcatacag tatgtaataa ttacgtcagg gtaaatgggg 20460 tatccatcat ttcaagcatt tatcctttct ttgtgttata aacaatccaa ttttaggttt 20520 tgttttgttt tgttttttgt ttttttgaga cggagtcttg ctctgtcacc aggctggagt 20580 gcagtggcgc gatctcagct tactgcaatc tctgcttcct ggattcaagc aattctcctg 20640 cctcagcctc ccaagtagct gggactacag gcacctgcca ccatacccag ctaatttttg 20700 tatttttagt agagatgggg tttcaccgta ttggccagga tggtctcaat ctcttgacct 20760 tgtgctctgc ccgcctcagc ctcccaaggt gctgggatta caggcgtgag ccaccacgcc 20820 tggccaaatt ttagttattt tcaaatgtag aataaatgtt ggctgtagtc aacctgttgt 20880 gcctatcaag tactagatct tatttattct atttttttgt gcccactaac catcctctct 20940 cccactaccc ttcccggcct ctggtaacca tcattctgct ctgtctccat gagttcagtt 21000 gttgtaattt ttagctctca caaataattg agaacatgtg aagtttctcc ttttgtgcct 21060 ggcttatttc acttaacata atgacctcca gttccatcca tgttgttgca aatgacagga 21120 tctcattctt tttctgtgtg tataaataca ttttctttat cctttcattc atctgttgat 21180 ggacatttag gttgcttcca aatcttagct attaagaata gtgctgcata caaaaattag 21240 ccaggcatgg tggtgcacac cgtaatccca gctactcagg aggctgaggc aggagaattg 21300

cttgaacctg ggaggcggag gttgcagtga gccaagattg caccatcgca ctccagcctg 21360 ggcgacaaga gcccaactcc gtctcaaaca aacaaaaaaa ggaatagtgc tgcagtaaat 21420 gtaggagtac agctatctct tcaatatact gatttccttt ttttggaggg gtatatacct 21480 agtagtgaga ttgctggatc atatggtagc tccattttta ggttttttga ggagccttcc 21540 aactgttttc cttagtgatt gtactaattt acattcccac caacagtgta tgagtgttcc 21600 cttttctcca catccttgcc tatcttttgg ataaaagctg tttttaactg gggtgagatg 21660 atatttcact gtagttttga tttgcatttc cccgatgatc agtgatggtt gagcattttt 21720 tcatatacct attggtcact ttgagaaatg tctattcaga tcttttgccc gttttttaaa 21780 aatcagatta tgagattctt ttcttacaga attgtttgag ccccttatac atttttgtta 21840 ttaatccctt gtcagatgga tagtttgcag atattttctc ctattctgtg ggttgtctct 21900 tcactttgtt gtttgctttg ctgtgcagct ttaaacttga tgtgatctca tttgtccatt 21960 ctcactttgg ctttggctgc ctgtgcttgt ggagtattat caagaaatct ttgcccagtc 22020 cagtgtcctg gagatcacat actattttta taaataaaat tttattggaa cacagtcaaa 22080 cccattcatt tacatacagt ctgcgactgt tttttttttt cctttttctt tctttttttt 22140 tttttttaag acaggctatt actctgttgc tgaggatgga gtgtggtggc acgatctcag 22200 ctcactgcaa cctctgccct ccgggtttaa gcgattcttc tgcctcagcc tcctgagtag 22260 cttggattgc aggcgcctgc caccacgcct ggctaatttt tgtattttta gtagagatga 22320 ggtttcgaca tgttggccag gctggtcttg aactcccacc tcaggtgatc catccgcctc 22380 ttccttccaa agtgttggga ttacaggtgt gagccaccac acctggcctc taaattgatt 22440 tttacttaca atgagcacat ttttgttaaa tttctcgcta ttggcaggag aagaataact 22500 gaagaaaggg gagcaattct gatccttcta aaggttcttc ttgcaacatg tcagaaagta 22560 tatttagcat aatgtttctt cttaaaggga agaccttccc taccttcctt attacccaca 22620 ttcccattct ctgttgttat tactgagcga tagcattgga taatagaagc attagtttct 22680 aagtcaaaca ggaactcagt tgcctcatat gtaaagtgat aatattatct aattcacagt 22740 gttgggatta aacaggagta catataggct gtaaaaatgg tagctgctgt ttatttttcc 22800 agttgcctgg aattgccttt tcatttgatg cattccagcg gttctcttgc tgcccactgc 22860 aaaaaattga taccacatga tttgagaaca agccttggaa aggatagaat aacttgttat 22920 acattttcat aggttgggat tttttttctt tatagaatct ttctagatct acttcgtggc 22980 aattaaaaat tacttattaa ttttcccaat ctcctatcct agataatata tccatctgaa 23040 agagaattat aagtcagtta ttttggggaa gcagcatagc atagtgagta aaaacatagg 23100 ctttcaagtc tgatctctta ggttcagctt cagctttgcc atttacttac tgactgtaat 23160 cttagacaag atgtttaacc tctgcatatc agttttctga tagggctgtt atgagggatt 23220 aaatgagata atatatgtaa agtgcccaat gtagtgccct gtgacatatt aagtaccata 23280 taaatatttg gttattaatg gtcatatgca tgtcatacaa atctgaatat gtaaaataaa 23340 tcagattgta gtatgaatgg atgttcaaaa aggtaaatgt agaaatttta ttaagactga 23400 aatatagcat gtgattttta ttttggtttt tattctttag gtattacatg aaacctttcc 23460 taaacataca tttctgatga acggcttaat tcaaggagta aaggtaggat cagtcataca 23520 tatatatgtg tatatataca tacatacaca tacacgcaca cggatgaaca tacataaata 23580 catatatata actatgcgaa tatatgtttg tttactgatt aataacttaa tttttataat 23640 tagatggagt atattttgag agataactta atactttcta gacttgagtt ttaaatatga 23700 tctaactaca atatagccaa tcatgcataa taataaacca catcataaca gattccctgt 23760 ttttatgttg gcattttaat tccagagatc tcaatgattt tgtaagacta cagattgaag 23820 aggaaaaaac tatactaaaa aacccccatc aatataaaac tgtatcagta gggtaaagag 23880 ttggttatta tatgaattct ttgctctttc tttttcgagt tttttttttt tttttttttt 23940 ttttttgaga cagagtcttg cactgtcacc caggctggag tgcaagtggc atgatctcag 24000 ctcactgctg agaacctctg cctcccaggt tcaagcgatt cttctgcctc agcctcctaa 24060 gtagctggga ccacagacat gtgccaccac acccggctaa ttttttgtat ttttagtaga 24120 gacagggttt tgccatgttg gccaggctgg tctcgaactc ctgacctaag tgatctgccc 24180 tcgacctccc aaagtgttga gattattggt gcaagtactg tgcccagctc aaattcttta 24240 ctcttgattc agtttgacaa acaagttttc gaaataggta attagcctgt tttatatata 24300 tataaatata tgttttatgt tttatatata tatatacaca cacacataca tatacacaca 24360 catacacaaa caggtaatta gcatatggaa ttgctattgt ggatttattg tgattgagaa 24420 tttattagag cagttcatat ttaataccta cctggagccc cacagatgat tctaaactat 24480 cttgggaaat tttaaattta tatatagata agcaattgtt tatttaaaag tttgtgatat 24540 atttacttta gaaacaaagt tgttgaaaat ttttctatag gagtaaaatg atttattttt 24600 ggttcatgct catagatgtg tggtattcat ttttttcatt taattttttt agggtttgtt 24660 gtcattcctt agtgccccgc ttattggtgc tctttctgat gtttggggcc gaaaatcctt 24720 cttgctgcta acggtgtttt tcacatgtgc cccaattcct ttaatgaaga tcagcccatg 24780 gtatgtgcac atttagatta tagcaactaa atatcacttt cagctattgt tttcttaatg 24840 ttcctttatt tctttcactt gtgccatctt ggttatgagg ttttaatttt atttttctaa 24900 tacatttatt ctttcacaca gataagaagg cctctaaaaa tacagtaggt aatagttcat 24960 aaagatttta gaaatagttg acactgttgg aggcatctag acttctggct aacttaattt 25020 tggattccag aacccaaatt tcaaaacaat attttgggga ctggatagga ttgctaactt 25080 tttttcttgt atagaatgtt aaaaacagac acaaaatttg tcattatcta tattagttag 25140 gaataggttt tgctatatat caaaacccaa aataagagtt gcttaaaaat caaatttctc 25200 ttttcatgtg aaaaagggtc tgcaagttga gcagtctgga gctggtatgg cagttccagt 25260 ggagcagact ttttctatct tgctgttctt ccatcctcat ctctcacctc actgaacaaa 25320 gtggcttctt gagctctagc catcaagtca tactactgat agcaaggtgg aaggagttgc 25380 taagaagagc acatcacctt cttttaagga aactttacag aattcccatc ccacctctgc 25440 ttgcatttct ttggtcagat ttcagggata tgctgctcct agttacaaga gtctggactc 25500 aaggccacct tgattaaaat cagtattttt tttttttcca ctcaggaggc aagggaagga 25560 aagctaacaa aaagggaata attgtcctaa agtcagagcc caatttggtt gtagatttat 25620 agatttattc tgactaatgt tctttttact ccactgagta ttaatgttag ttctacatca 25680 cggatgggct ttatttccat ggtgttatct acaaaggccc aaaggtttat atggggcagg 25740 atcttcttct tgtagatgag atacaattct aatagaccaa gcctttgatg aaggccactc 25800 atgcacatga aatcttacca aaaattgaac atggaatgaa caaagaattt gctgtgttaa 25860 gcagtgtgtt agaaaaattt tagtagtgat agtcttggtg gaatgatatt ttgaaagctc 25920 attatgatta tatgtgattt tcagaaacta aacatcagta ttacaataga aacttcttat 25980 tcccagctat tttggaatta tttatagcaa aatatagttt actctttaaa tattttgttc 26040 actgtttata gtaactgtct attctcagtt ttatgaacac tgaatcctgc catataggtt 26100 ttttgtgtgt aaaactgagt tatttgtttt gccagcattt gaaaagctaa agataactta 26160 tggaaaacat ttagttacta tatagaggct caaaataaat atgaagaaac ttgttcctca 26220 gtcttgttta ctggattttg ttttttatct agtgttgtgg tgaaaaacag taatggatgg 26280 attataatag actttagttt gttgctgttt ttggcagaaa aagcaaaatc acttaacagt 26340 tcaacagttg gtcaaaagat ttgcctgaat acatgatcta attttaagta ttcctgtata 26400 gtagctgagc tgctattgaa gggctccttc tagccctatg ttttcataat atctgtggct 26460 tatatttatg gaatagttaa tccatgaact atcctagtaa gctgttgact gaaatgagct 26520 gctcttacgc ttaattaact tataaaaatg aaagaagatt aaaacaatgg taattgctct 26580 aaccatttct tgttatcttt cattcctagg tggtactttg ctgttatctc tgtttctggg 26640 gtttttgcag tgactttttc tgtggtattt gcatacgtag cagatataac ccaagagcat 26700 gaaagaagta tggcttatgg actggtatgt atgtttattc tatacctttt gtatctgctg 26760 agaaatgcct tgtttttaag ataaatatta ttataaggag tgcaaacctt tgcattacaa 26820 gatttttgcg taaaatatat tttgtaataa aatcattcat tagactacat ttaaaatttt 26880 tttgcggtat gaggctatgt aagttttgat tcttttcatt tagtagatat tcataagtca 26940 catgtcagaa ttgaaattat agtatatttt accttgtaga gttcttttta acagaatcct 27000 aaaaataaga attatttagt atgtcaagag ttaaaaaaaa tcactactca tttaatgtct 27060 aatctaaaat acaacaggct aacatctagc tcagggatca gcaaaccttt tttgtggctt 27120 tgtgggccac gtacagtctc tgtctcattc ttttgttttt gcatgtgtat ttatgtttat 27180 aaactcttta aaaatgtaag aaacagccag atttgagcca tagtggtagg tcgccaactc 27240 ctggacactg ttttggtaaa ctaaattatg gcagtataat gtgtcatcta tcaaatctag 27300 gaattaaagg aaaaaagcct agtaatagaa tgactactat aggcacaata atagatcact 27360 actgaatagc cagaaatagg acagtgatgc atttcggtaa atgtgagaca aataccttgt 27420 gataaataag gactgaatat tgtgttgggc tgaattagtt ttaaaaggga ctgatttctg 27480 attcaaagga cgttatagtg aagaatcata agatttttgg ggaggaaaca cctatagaga 27540 gaaagttaga aaaagaacta ataatttctg gcctgttcag tggctcacac ctgtaatctc 27600 agcactttgg gaggttgagg caggcggatc acttgagatc aggagttcac gaccagcctg 27660 gccaacatgg tgaaaccttg tctctattta aaaaaaaaaa aaaaaaaaaa gtgaaaagaa 27720 aaagaactaa tgatttcagt tgtaaacttg gaacattaaa tgatacaagg ctgatgatag 27780 ccaggatatt taaaaaatag tctaattaag ctatagttta cataccataa aatttatcct 27840 ttttatgagt atagttcagt gaattttagt aaatttatac tgttatgcaa acaccaccat 27900 aacccaattt ggggttggtc ggttggttgg ttggttcgtt ggtttggttt ttttgacgta 27960 atttattttc ccatagccaa agttttgaaa ttaacaattt tcaatctgga ggttctgtgt 28020 attaagccat gttctggcaa aaaacaaaac aaaacaaaac aaaacaaaac aaaacaaaaa 28080 acactgaaat cttctagaaa taatatggat gcagaaaaaa ggtggggaag tggccaggca 28140 cagtggcatg tgcctgtaat accaccagtt tgggaggcca aggcaggggg attgcttgag 28200 gccaggagtt tgaggctgca gctatgatca tgccactaca ctccagtcta gggtacagag 28260 tgagaccctg tctcttaaaa aaaaaagttg gaggggccag gtgcagtggc ttataatccc 28320 agcactttgg gaggctgagg caggaggatt gtttgagccc aggagtttga gactagcctg 28380 ggcaacatag tgagacccca tctatacagg aactttaaaa attagccagg tgtggtggtg 28440 tgtgcctgta gtcccagcta cctgggaggc ttaggtgaga ggatcacttg ggcctgagag 28500 gttgaggctg cagtgagccg tgatcgcacc actgcgctcc aacatgagcc acagagcgag 28560 acctgtctcc aaaaaagggg gttggggggt gcggggtgac ccctgtgatc ttttttctga 28620 gcagaaagaa atggctacca agtggagaga actgaggaga agggaaatga catgaaacaa 28680 ctgtactgac ttgctcactg tgtcacaaat gtgatctctg taaatgccct caaatgtctt 28740 cagtgaccct catagtgaga accattttcc ctttccccac acttgtgcca gagccctgct 28800 gagatctggg tccctctgaa accacaccta gggctgcaat aacaaaataa ccactacatt 28860 tgaaaatata tatttatatg tatgtgtgtg tgtgtatgta tgtgtgtgta tatatatata 28920 gtttgttttt tgttgagacg gagtctcgct ctgtcaccca ggctggagtg cagaggtgtg 28980 atcttggctt actgcaacct ccgcctcctg ggttcaaacg attctgctgc ctcagcctcc 29040 ccagtagcta tgcccaccac catgcccagc taatttttgt atttttagta gagacggggt 29100 ttcaccatat tggccagtct tgtcttgaac tcctgacctt tggtccgcct gcctcggcct 29160 cccaaagtgt tgggattata ggcgtgagcc atggcgcctg gcccccatgt gaatatatta 29220 aataccattt aaaaaaccac cacaacccag ttatagaaca tttccatcag cccaaaatgt 29280 tccctcagcc ctgtttgcca tctgtcccca tgctccacct gtgaccccaa gcaaccaaca 29340 atttagcttc tgtcaccatg gttttgcctt ttctagaaac ttcatagaaa ttaaataata 29400 caaaacatct tttgtgtcta acttctttca cttggcataa tcttttgaga ttgatccatg 29460 ttgatactat agatcaatag gttctatttt tgtctctttt cctttttttt ttttttgaga 29520 cagggtcctg ctctatcccc caggctggag tgcagtggca tgatcatggc tcactgaagc 29580 cttggcttcc tgggctcaag cgatccttct gcctcagcct ccaaagcagt tgggaccaca 29640 ggcatgatcc accatgccca gctaattttt ttctttttga gacagggtct cactctattg 29700 cccaggctgg agtgcagtgg tgccgttaca gctcactgca gcctctgtct cccctctacc 29760 tccctgcctc aagtgatcct tccacctcag cctcccgagt agctgggact actaattttt 29820 gtatgttttg tagtgatgga gtttcaccat gttgcccagg atggtctcaa actcttaaac 29880 tcaagtgatc tgcctgtctc agcctcccaa agtgctggaa ttacaggcat gagacactgc 29940 acctggccag tagttttttt tgattgctgt gtagtgtatt cttatccatc agttgatgga 30000 catttgattg atagctagat gtttgaaatt actagaattt tatgtacttg ttcaaataat 30060 tgacctttga aaattgaatt gcttgcctta agcaatagag ttgcaagtaa gcattcttgt 30120 gaagtttaag ttctccatcc aaaagtcaaa aatggcatag aaacagaata aaattccaac 30180 attaatctct atgctttgaa agaatatggt ccttttcctt tccttccctt cccctttcct 30240 ttcctttgcc cttctcttcc ccctcccctc ccctcccctt tccacttttc actttcactt 30300 tcccctttcc ttttcgcttt cacctttctc ctttcctttc cttttctctt ttcccttccc 30360 ttcccttccc ttcccctttt ccctttcctt tcccctcccc ttttcccttt cccttctttt 30420 ttctttttct ttccttttcc tttcccttcc cctttcccct tttccttaag ccttttccct 30480 aagccttttc ccttttttaa ccctttcctt ccccttttct cttccccttt ccccttttcc 30540 tttctcctcc tctcctttcc tttcctcttc tcttctctcc tctcctcttc tttccccttt 30600 ctccattcct tttccctttg ctttcctttc ctctttcctt tccagacagg gtgttgccca 30660 gactggactc actcttggga tcaagtgatc tcccacctca gcctcctgag tagctgggac 30720 tataggcagg tgccacctca cctgactaag agtgctattt ttatgaagtg tttcctgctg 30780 tcacatctgc taatttgtag gctgttgtcc agtaggctag aaatgtctgc ggttaacagg 30840 tttgctctac tcgtgtcctt ttcaacttta atcttcatct tcaccaggct taaaaaaata 30900 gacttcctca gagttttaga gatgttctta atttatctgt gatttcattc ttcctaaccc 30960 tgccaactaa aaagattacc aagctcagtt ttgttccagg gcttaacata ttattcatga 31020 gaacaggaac ctccaagtct ttaagcttta tttcagctag cccttcagta tgtatcaaga 31080 taaacgttca tttaatttta atattggaaa agtcacagtg aaattggatt tccttagagc 31140 agtggatttt agactccttt cacagagagc acttaagggt ttatggagat gcccttaacc 31200 aagcttgtcc aacccatggt ccacaggctg cacacatggc ccaacagaaa ttcataaagt 31260 ttcttaaaac attatgcagt ttttttttct ttaagctcat cagctattgt tagtgtattt 31320 tatgtgtggc ccaagaccgt tcttccagcg tggcccaggg aagccaaaag attagacacc 31380 cctcccctaa ggaccagcat gactggcagt caaggagggg tgtttgtaca gtgcccaggc 31440 tctcaaccct tcctcaacta aaagagttaa aaaatttaaa taggccgggc atggtggctc 31500 acgcctgtaa tcccagcact ttgggaggcc gaggcgggcg gatcacgagg tcaggagatc 31560 gagaccatct tggctaacac gggggaaacc ccatctctac taaaaataca aaaaattagc 31620 cgggcgaggt ggcgggcgcc tgtagtccca gctattcggg aggctgaggc aggagaatgg 31680 cgtaaacccc gggggcggag cctgcagtga gccgagatcg cgccactgca ctccagcctg 31740 ggcgacagag cgagactccg tctcaaaaaa aaaaaaaaaa aaaaaaaaaa tttaaataga 31800 ggcagggtct tgctgtgttg cccaggctgg tcacaaactt ctggcttcac gcagtcctcc 31860 caccttggcc tccccaagtg ctgagattac aggcatgaac catcacaccc agtcttctta 31920 aaaaaatctc ttttacctat gaatttgcca gtaggattta ttggaacaga gggctccaag 31980 gcttagaaag tttgaagaca gtgtcctgag aggctatcat ttattttatt ttatttttga 32040 gatgcggtct cactctgtca ccctggctgg agtgcagtgg tgctgtcatg gctcactgca 32100 acctccgcct ttctggctca aaggaattct gccacctcag cctctgaagt agctgagact 32160 acaggtgcac accagcatgc ccagctaatt tttctttttt cttttttgat acagacaggg 32220 tttctccatg ttgtccaggc tgtttttaag gcaagaatct aattctttac ttttcctgcc 32280 aaaggagaga gtataagaaa agtggggcca ggcttggtgt ctcatacctg tactcccagc 32340 ccttcgggag gctgaggtgg gaggatcgct tgagctcagg agttcgagac tagcctaggc 32400 aacatagcgt gacttccacc tctataaaaa ataaacaaaa ttagctgggc gtggtggtgt 32460 gtgcctgtag ccccatcagg agatcttcag gcaggaagat ctacttgagc ctgagaggtc 32520 aagactacag tgagccgtga tggcaccact gcactccagc ctgggcgaca gagcaagacc 32580 cagttccccc actctcgccc ccacaagaaa aaaagataaa tggcacaggt aggaagagaa 32640 aagggagggt gtgcaacaga aggcctgaca taaatcaaga ttatgaaagg agttatgtgg 32700 tgttgaggaa aaaagtagcc tgactaatct ctgtctatcc ttaatttatt gcaggtttca 32760 gcaacatttg ctgcaagttt agtcaccagt cctgcaattg gagcttatct tggacgagta 32820 tatggggaca gcttggtggt ggtcttagct acagcaatag ctttgctaga tatttgtttt 32880 atccttgttg ctgtgccaga gtcgttgcct gagaaaatgc ggccagcatc ctggggagca 32940 cccatttcct gggaacaagc tgaccctttt gcggtaagtt tatacttttt ccttctcctt 33000 gataaaaaag tgcatgattc agtgcagcat taatattttg ttgtggatat ttctttaggg 33060 aaaacatctt gggtttttct tttaacattt tgaaatactt ttcagaatag tttggggaat 33120 atgaaataaa taaaaaggac aactagttgt ccatgagtac actgccaaaa ggaatctctg 33180 catattctta agaatagttc agtggttttg atagaaaacc ttatatcaat cagtcttttt 33240 cttgttctag tccttaaaaa aagtcggcca agattccata gtgctgctga tctgcattac 33300 agtgtttctc tcctacctac cggaggcagg ccaatattcc agcttttttt tatacctcag 33360 acaggtaaaa tcctcttcca ctaaggtgga cttttctttc attgtctagt gctttaataa 33420 aaatatttaa tcttgagaga actgtaatag aagtggcctt taaaaatgaa tatcattgga 33480 cttggtatag tggctcacgc ctgtaatctc agcactttgg taggccaagg tgggtggatc 33540 agctgaggtc aggagatcaa gaccagcctg gccaacatgg caaaatccta tgcttactaa 33600 aaatacaaca actagccagg cgtagtggcg ggcacctgta atcccagcta cttgggaggc 33660 tgaggcagga gaatcacttg aacccaggag gcggaggctg cagtgagcca agattgcgcc 33720 attcactcca gcctgggtaa caagagcgaa actccgtctc aaaattaaat aaataaataa 33780 ataaataaaa agaatattat ttggtctact agactttacc tcctattctg tgtggctgat 33840 gttccttatg catttctaat gggggttatt tggtatatta agatatttgg cttagggaga 33900 aagatagttt tccctgtcca tataggtggt ttgagtttgt tggctataga attgatggga 33960 tgatttaacc ccttcacctg ctccagcttc tttgtgattt agagcatatg taagtagagc 34020 agctagccaa aatgagagca aaaacaagta ttttcctcct tgacactagt ctcactagac 34080 ggagatcaag cctttaacca atacatgtaa aatgcacaaa atactgcaat atttatttgt 34140 aaaatgattc tgagttcttg ataagtatct ccaatttagt atatccacat tgagggaccc 34200 accatggata agtaggcatt tttagtatgt taagatatgt tgtatttcct ctgaggaatt 34260 ctttgtttat aaatgaatta catttatttt tttctggccc attaaatgtt aatatacaag 34320 tagctgcagt ggttttctat ctggtataca tttgtattca ttaatgttac cttttctgga 34380 aacccttcta gataatgaaa ttttcaccag aaagtgttgc agcgtttata gcagtccttg 34440 gcattctttc cattattgca caggtgagtt tcttttttta gttagagtga tgtcagtgac 34500 cctggctggc catccaaact ggggcctcat ctagtgatgg tatcttggtg gaatcaacaa 34560 agtcaggagt ctgaggttga taggttcaga aattcaattt gtttgtgtag ctagtgttga 34620 tcagattttt gggactgaca catttaatat aggatatata aacgtaaaag ctagtttatt 34680 gatgtatact aggatatatg tctggatgaa gttagaagtt tcatgtcttg tagtccttgt 34740 cctatttatt cactcattca acaaatattt attgaatacc taccgtgtgt caggcactgg 34800 ggatacagca gtgaacttaa ctaaattctt gcttttgtag agtttacatt ctggtgggat 34860 aagacagaaa ataaaaacac caatgtaaac atatcagatg tattgagaac tacagagaaa 34920 aagctaaggg tcatatggag agtgacttgg gagaatggtc ttaagggcac agggggcatg 34980 gttgagaaga ccttgctaat gcaaggtgac atttcagcag agacgcaaag gtatgaggaa 35040 caagctgtag gattatctgg aagaagagaa ttttaaacag tacaaacaaa attctcagga 35100 gagactgtag ctggcttgtg taagaaacaa cagggggcca gtgcggctcc agttgagtga 35160 acagttgaga gcatcttaga aactgaagtc caagaggtag tgagggacca gatcgtgcgg 35220 ggcttacagg tcctcataag acttgagtaa gataggaagt cattggtggg ttttgagcag 35280 cagaatgaca tgaaagtgtg tatcttggca gcccagttgg taaacagttg ttgtgtgatg 35340 aataagatat taatcaacac aggaatttgg attttctgag gagatatttc atcctggctt 35400 ccaaactgtt tctgtttaac aataagaata ctatcttttt tccagtgacc accataattc 35460 tctcacatca cagaacattg tcctccttcc tgtgaaaaag ccccaccccc tttcttgcta 35520 tttggctttc tgtttctaag acactatgaa gacaatctag tctaagttaa tactttttct 35580 caccttagat gtaatctact agattacaac ttagttttat tctagggtat ttttaattga 35640 ctcttggatg gttcaccatt tctcgagtag gattccttct gcaggtctca tgattcattc 35700 tgttttggtg agtttagcaa caaatttcaa atttaaatcc tatatgcctc cccaagcctc 35760 tgcacataca tatacttggt gttgagtatt agtactattt ctagcttcag gtttgtccta 35820 aaaatcatca gtctggaaaa acaatgcatt taaatattca ttcctagcca tgagaaaagt 35880 gctttttaac tttggaggaa aatatactgt agcctttata taaaaatggc tttaaaaaaa 35940 gtttttgagg ccaggtgcgg tggttcatgt ctgtattccc agcactttgg gtggccaagg 36000 tcagggaccg cttgagccca ggagttcaag accagctcaa gcaacttggc aaaaccccat 36060 ctctaccaaa aaaaaaaaaa aaaaaaaaaa aaagaaagaa agcccggtgt ggtggtgtgt 36120 gcctgtagtc ccagctactc aggaagctga gatgggagga ttgcttgatc ctgggaggtc 36180 gagggtgcag tgagccacag ttgtcccatg gtactccagt atgggcaaca gaatgagacc 36240 ctgtctcaaa aaaaaagtgt aaggaaaata cacagttagt atgtgtagaa cttgatgaat 36300 tatcaaagat taacccaacc ttgcaataga tgtgatcaac ctgggcatga gtatcttttt 36360

catacattgc caacattaga tttgctaaca ttttgtttaa gatttagatg aagagagcag 36420 actaatactg taatgacaca taaaagattg atagctataa ggtcttaagt tctgtttctt 36480 acttaaatga ccatgggagc tgtatgcatc taataaatgt agtcagtgac actgcagacc 36540 cagtgatgag tggagggtgc ttttgagggt atttttcctc tgtttagcag atggcatctg 36600 gcactaggtt acaagatgaa aaacagtctc tgagagtgca gaatctggca gggcagacag 36660 gtaaaggaga ggaatgtagt tggatttgta ttggaaagat cagtgcagca gcagagccct 36720 gaaggcagta agaccgcaag gtgcaagcaa ccctccaaac accattgctg acacagcatg 36780 acacacacag tccatgtagg aacaaggtcc ttaagtgacc atttaggttt tggtgcttat 36840 gtagaagtaa gaattaacac tttatatcat atatgagcta ctacattatt acatgtatag 36900 cctcaaaggt agaagaatga tcttgtattc tcatttatgc agaaatatac aattgagaat 36960 gacttgtccc ctgttgacct tccataccac tgaaagacca gtgtatttct ccctcctctc 37020 cagcagcacg gagctgctaa tagctctcca ttaatgcatg tttgctttat ttcttaccca 37080 cgtgcttttg ttcctgctat tctttctgcc tccccttcct ccaccctaag tagccctttt 37140 ctgggtcccc atgctcatgt gcgcatatct ccatcattgt acagaataca gtgtagtaga 37200 gattttatct ctgtttattg ccttagtttg tgagctctag cggacctctg agtagatgat 37260 gaggtcagga ttatatcata ttcatttttg tcaccctagc accctgaact gccaggaggt 37320 gctaaactaa aggtcattgg tttttttcca taatgttaaa aaaaaaaaaa acttaaaaaa 37380 ttagtagtaa gcactttaaa attgggaagt gttacgtgaa attatagtta tgtagcttct 37440 cccccaaaat gattagatct ggtaaccggg tgtgggccaa ccttccagcg agagccaagt 37500 agttgctgtc ccctttggaa ccttttcttt ttggtttatc atcagcccca ttacttcctg 37560 ggcaccggta cgtataagag ttcctaacac ttgcactaag taagtgttta catgagaaca 37620 tcaatatagt tctacacatt tcttttttct cagtgttttc ctatccagcc ctctctgtgg 37680 gggtgactcc cacacttact cttccagtcc agtccctgag ctcctatatg acacattggc 37740 tgggtatctc agccttaaca tggccaaaat taaaatctgg gttccatccc ttgcccgcca 37800 cccctatgct cctcatctca ttcagtggct tcaccaccgc caggttttgg gggccagaag 37860 ccttagcgac attcatgaat cctttctccc taaccttaca ttcagcccat caaatgatac 37920 ttcctatcac ctctctctcc aaaatatatc ttgaatcaga gcgtttctga tattctccat 37980 tagtaggaac ccaatctgag ccgtggccat atcttccatc tggtctctct gcttccatct 38040 tgcctcacta tagttcattc tgcacttggc agagtaattt ttgtaaaatg gaaatttaat 38100 cgcatcttac ctataactca ctttcctttg caaccagaat aaaatctaga ctccttatta 38160 tgtcattttc ctccactctc cacatggttg agtatgttct gtttcagtcc agggcctttg 38220 cacttgctgt cagccttgct tggggtgttc tctttcccca gatctttgca taactaggtc 38280 tctcccctta gtgagctctc acttcaaaag gccttccctg gatcctggtt taagcagcat 38340 ctccatcaca ctgccctgtt ttattttgtt catagcagct acaacctgga atatcttgtt 38400 aaataagcag gctcatatgc atctttcctc catgagaagg tagtccgtgt gttgatggat 38460 actttgactt actcacccat gcgtcttcag gccaagaata agagttggtc ttattaggaa 38520 tttggcaaat atttgttgac agactaacca aagctgatgt tagtgttagc ttagctgttc 38580 atcagctatg ccatcttgct taagtcattt aacctttggg actcagtgct gtcatcttca 38640 aaatgagagt taaattaagg gacaccaaaa attttttccg ttcaagaaca cttatatatt 38700 aagtattttg ttccattatt attaatatta ttgagaatat tataacattt agaattatgc 38760 atgctttcca taaacactta ttaagaactt actgggtgct ggggatataa atgtaaataa 38820 gagaaaagtt cctgccttca agaagaaaat cagtgttccc agttacagtg ttgtaagtat 38880 cggtgtcttt tagggctttg ggtcacaaga taggctgtag gggaaggagt ccaagaggag 38940 gttctcacag cagtgggcct gctgcagtaa ttctgcacat aggacgttac cccaggaaag 39000 ggggattggt gtatctcatt gtttccaatt ttaatgactc atctcatggc cttaagaaaa 39060 tatattcttg gccgggcacg gtggcccaca cctataacct tagcactttg ggaggccaag 39120 gtgggtgaat cacctgaggt caggagttcg agaccagcct ggccaacatg gtgaaacctc 39180 atctctacta aaaatgcaaa aaaaatttag ctgggcatgg tggcacgtgc ctgtaatccc 39240 agctactcgg gaggctgagg caggagaatc acttgaacct gggaggtgga ggttgcagtg 39300 agccagcatt gcgccacagc actccagcct gggcaacaag agcgaaactc catctcgggg 39360 ggaaaaagaa aggctgggca cgtggtggct cacgcctgag ataccagcac tttgggaggc 39420 cgaggcagct ggatcacaag gtcagaagtt cgaaaccagc ctggccaata tggtggaacc 39480 ctgtctttac taaaaataca aaaattagca ggcatggtgg tgggcacctg agtcccagct 39540 actcgggagg ctgaggcaga aaaatcgctt gaaccccgga ggcagaggtg gcagtgagcc 39600 aagattgtga cactgcactc tagccttggc aacagagcga gactccgtct caaaagaaaa 39660 aaaaacccaa aagaaaaaga aaatatattc tccagttaat cttatctata aaaaggaaat 39720 gaggctaata atgcattcta agcctttttt attgaattgg gatttattct ttgagaacag 39780 ctttccacaa aggggaagat agtcatttct gcagataagt acttactggc tagatgggtt 39840 ggttgaaggg ctatgagatg accgcatttt ataagtactt tctggtaata ttaatgatct 39900 ctgcttgaga agtgtcagct ttcttagact agcattcctg aaagaacacg tgctccaggg 39960 tacatggctg gcccatgcca tcctgttccc actgagctgg gagttgatgc tcagccttct 40020 tcactgtcct gtctcttggc tgaagtgcca gggatttcat cattaagtga aatctatttt 40080 ttaacagcat ttcttccttg taatgcttat catcatctca atgaatgatg agaacaaatg 40140 ttttctgctc tgtgagttcc tagagccata taaagaatca cagcttttta gatgaaagtg 40200 ctaccttccg ggatgttctt aaaagtagtt tcccagaagt tcttcaactt tgcattatag 40260 ttccctgttc ttctcagaca gttggaggcc acagagcttt gggtatactt acagtttcct 40320 ttttcataat taattgaaag ccagtctctg atatagtcca caaataaaat catttttaat 40380 tatttctaac cctaattaga atcctaatca tttctaatct ttctgattat tttttataat 40440 attggccacc agttcccacc cacaagtcat gggtgacttg agatggtctc tgtcacccag 40500 gctggagtgc agtggtgtga tcatggctta ctgcagcctc gacctcctgg gctcgagtga 40560 tcctctggac tcagcctcct gagtagctgg gaccacaggt gtgtgccacc agcctggcta 40620 atttttcaat tttttgtaga gatgtggtct ctttatgttg tccaggctgg tctcaaactc 40680 ctgggttcaa gcagtccttc catctcagtt ccccaaagtg ctgggattac agatatgagt 40740 cactgtgcct ggcctaaatg tttctttcag ttgaagtttt tctcagctaa ctgctggctc 40800 tgggcaagtt tccttttctg gtttggttgc cgaagaatat aattctacat ggaactcagt 40860 cttatactca cgtgatttaa gtgaaagtct tagacaagaa agctgtgagt tctaattgct 40920 caaaaatcct tgaatgaatc atttgctttt cactggcata tttgtgatta aattcttaag 40980 tggccatctc aatttaaaga attcaaaact tatattttat gagttttaaa gtgtcagccc 41040 atcataaaat agtgatttcc tgaattattt ttacttgtct atggacttac tagctatctt 41100 atgtagtata ttaaaaacgt tagttagaat agcaaaaaaa aattttctaa tgttcccaaa 41160 tcactgctga actttgttga ctttgaaaga aaaaggaggc acaagaaaac ccacccactg 41220 ataatattgt ttattaaccg ctgattattg ccaggcacaa ttctaagaac tttatgtaaa 41280 tatatctcat ttaattccca tgacaaggtt ttgaaatagg tgctgttatc cccatttgca 41340 aagagacaaa agtgaggctc aaagtgaagt gacttgctga gagccacaca aagccaagat 41400 tatgatccag gctgtttttc taaagtctgc tgtatagtat actgcattta tcccaaatca 41460 agcttattaa tttactgttt ataaaaggca tcatggtttc aacaacagat taacttaggt 41520 aaataatata tgggtaatca ttgttctgtt agtttttctt tagttgtgga aataagcatt 41580 ttagactaac ttggacctaa acaagcttta aggctattat gtaatgggga tctccaaatc 41640 attagttaga acttttgacc cttccatttt caactactga tttaagtggt cctcagtaga 41700 aatgtactga ataggaagtt ttatctttca gttttctaac tctcagtctg gatctatgtc 41760 agcagaggga cttttcatct gcttatgtga cctggactag tgatctcaga catattcagg 41820 gcaattattg ctgaaaatca gccaaattgt agaaaagtgc caatagtcct tttatagtgt 41880 agattgaaag aagtcacttt ttaaaacttt attctgataa atcttttttt ttttttttca 41940 gaccatagtc ttgagtttac ttatgaggtc aattggaaat aagaacacca ttttactggg 42000 tctaggattt caaatattac agttggcatg gtatggcttt ggttcagaac cttggtaagt 42060 ataaatattt taatgttaat atttttaatt ttggtgttag cccttgtgtt tttatttgct 42120 tctcaactga ggggtagact gtaatctgtc tcatactatg ctttttatct ttcaaaatgt 42180 gtctaatata agtctgccac ttgtatattt atatgttctc ctagaatggc ttgaggatta 42240 aaaaggtgac cttttatagc taaatgacag gctgaatttt tgaatgagat tatacagctt 42300 ttgaatcttt aaggagcatt taatctaaat cagtccgtta ctaggaaaaa gtatgtaatc 42360 ccatagcaac aaggccctga aagttattta cattttttgt ttttctggtc aggaaaaaga 42420 aagttgtata accagtgatc attactgata gcaaaccaga agtgaaaaca atccagttat 42480 tttggtgcca gcttcatgct gtgtctcagc ttttctgacc agtcgggttt cctgcagggg 42540 acttgagtag tgacgcttgt tgtcaggctg cccacgtaga tagtattatt gtgctcaggc 42600 attatggcac tgaactccat ggtttgcact aatatttcaa acaactgact gcctcgtctc 42660 tgtttggact tggatataag caagcatcaa gagggtgatg atttgttctc caaactagcc 42720 tttgcaaaga ggtgctcaca attgaaatta cctaaaacat ttcttttaaa catcaagcca 42780 ggaacatcca agttacttgt tctttacaat ttaaggatta gatcaaatca gcgatatctt 42840 cacaaatcca tccataagaa ctttgccaaa gacttgttgg cttcatgtgt ttggaaatat 42900 ttgatgatgt ttcgtcatct atattataca ttatcctcaa tataacctct caattgcctg 42960 tagaaataat acccagcaca ttttacagtt tggaacatga tatccctatt ttacattaca 43020 ccctcacagc actcttgtga attaagttgc tgtctttgta tacagggtca gattgttaag 43080 cgacttgccc atagtcacct agtaagcaag ttcagttctc cttttctcta cagccatttc 43140 acagtaagaa ttacttaatt atgtagtttg actttcaggt acagtggaga agaatttact 43200 gtttttgttt tgctgctctc cttataggat gatgtgggct gctggggcag tagcagccat 43260 gtctagcatc acctttcctg ctgtcagtgc acttgtttca cgaactgctg atgctgatca 43320 acagggtgag ttgataggaa ctagcgataa ttatttaaaa gtacagaatg ttctaatcct 43380 gtgttctgtc tcctatgtac tgaaacataa gtatatcttc agggtagaga cttttaaaat 43440 tgcttttgat ataaacagga aaagcagatt ctagggtatt tatccttagg tagatacata 43500 ttcccttttc tctcacttag aatatgtggc ttatctgttc tgttcataga aaatttactg 43560 atgaggctgg gcatggtggc tcacacctgt aatcccaaca ctttgagagg ccgaggcagg 43620 cagattgctt gagttcagga gttggagacc agcctgggca acatggggaa accccatcac 43680 taaaatgcaa aaattagctg ggcagggtgg cgcatgcttg tagtcccagt tacttgggag 43740 gctgaggttg gaggatcact tgagtctcag aggcggaggt tgcactgagc caaaatcagg 43800 ccagtgaact ccaacctggg cgacacagtg agagaccttg agagacctgt ctcaaaaaaa 43860 tttactgatg aatgttggcc acagaatatt aactaagcat taagtttttg ctgtgttgtg 43920 ctagacacct tgtgggatat attcatagac cttttatgaa tgttgtccca gccacttggg 43980 aagctgaggc aggaggatta cttgagctca agagtttgaa gctagcttag gcaacacagc 44040 aagactccca tctctaataa aaaaaaaaaa agaaatcata tagtttcagt catctgaaga 44100 tatgtaacac agcaacatct ttaatgtcat atgctctctt tttttttttt ggtgggaaca 44160 tttaattctt gggatgctaa cagatgacaa atacttcttt gaaaaggata tgttttgtct 44220 agtcataagg ataaaagggg cactgcaaaa cagtggcagt tgtcacttgg ttgataaatg 44280 aggaaggaaa ggcctatggc caaagcaagt tgcattttaa attaaagatc caaaagagag 44340 aaacaaaaat caaatgctgt taaaacagtg ttattggccg ggcgcagtgg ctcatgcctg 44400 taatctcagt gctttgggag gccaaggctg gtggatcacc taaggtcagg agttcgagat 44460 cagcctggcc aacatggtga aaccctgtct ctactaaaaa aatcaaccag gcgtcgtggt 44520 gcacacctgt aatcccagct actcgggagg ctgaggcagg agaatcactt gaaccctgga 44580 agcggagttt gcagtgagcc gagatcgtgc cactgcactc cagcctgggc aacaagagtg 44640 aaactccgtc tcaaaaaaaa aagtgtgatt ttggctgggc acagtggctc aatgcctgta 44700 atcacagcac tttggtaggc tgaggcagga ggatcacttg agttgagttc aacaccaccc 44760 taggcaacaa agtgagaccc catctctaca aaaagtacaa aaattagcca ggtatttgag 44820 cccaggaagt tgaggctgca gtgagccaag tttgcgccac tgcactcccg ccagttgacc 44880 cccccacttt atttttgact aaaaggggtc ttaagtactt gcttggggac agataaattt 44940 taacatttgt agttgtgaaa ttgtgactga tttttaacca gccatgtttt gaaggctgta 45000 atctagggaa ataaagtagt tttgcccact tgtttctatg tgagacctat atatgggtac 45060 atcagagctc atgtttgcat aggacagctt actacacttt gtagagctga tagcttctaa 45120 atattaatag tttttaatga cattgctata aattgctagt gtctgataag aagcttgatt 45180 ttagattaag tgatttcata tttgtactgg ttctaaggta gggaaaaaaa accaggtagt 45240 ttactaagtg ataatttgtt taaacaatgt aggtgtcgtt caaggaatga taacaggaat 45300 tcgaggatta tgcaatggtc tgggaccggc cctctatgga ttcattttct acatattcca 45360 tgtggaactt aaagaactgc caataacagg aacagacttg ggaacaaaca caagccctca 45420 gcaccacttt gaacaggtaa ttctcattca acatgatcaa attgtatggt tcatttggct 45480 agattaaagg ctactggttt ttggtcatga aagcttttat gtgagattct atttgagatt 45540 ggataaaatg cttaaaaacc agagtttaag ggactgtgtt cttctacata ccaccttgtg 45600 aaaattggct gtgcatattt tttttccact tgagaatgaa aaattttaag tacctagttt 45660 gtcaatggca tatgtaacaa accatttctc tttactacta gtctcttttg aaacttttct 45720 aatatcacag ttgtgtatgt taactaactt ttcatacaaa aggcaggctt atggtaaata 45780 acctttgttc actgtttgct atatttccct cttttcaact gaaaattaat gccaaacaat 45840 gctgatcatt ttggccaata ttagcagctc atcagtttcc tggcttattt agcagagttc 45900 tgtacttatc ttccaaccca ctaagactac ttttaaataa aagaatgttt gtgggctata 45960 cacaaaactg gcagtgcaag cttctgctaa gaatgaatgt aattttgggc taaaacaaag 46020 gtctcttttt cttggtaacc tgtgtttttc tcacttccag tcacttgaaa ttataattac 46080 cttcttgaaa caaagaaatg gtaatttatt ttgctactgt agtaaatact gtatgctacc 46140 tgattttatt ttgttttttg ttgtttctga gacagtctta ctctgtcaca caggctggaa 46200 tgcagtggca caatcttggc tctctgcaac ctctatctcc caagttcaag cgattctttt 46260 aacatcagcc tcccgggtag ctgggattac aggcatatgc caccatgacc ggctaatttt 46320 ttttgtattt ttagtagaga cgaggtttca ccatgttggc caggctggtc tcgaactcct 46380 gacctcaagt gatctgcctg cctcagcctc ccaaagtgct gggattacag gcttgagcca 46440 acacgtgcag cctgatttta tttttgagat ctttctataa acgttttccc cttggactaa 46500 caaattaatc atagaatagc tgtgttcaca ttttgtgctg aaagtaatga tgtaatattt 46560 tcgcataggc tgttttgcca gttgttattc ccagaatttt atagagaatg tgatagtatc 46620 tcttttctcc taaaaaggga atgcctttta taattatggc tttaataaag agaagagcaa 46680 ttagcttgta attcataagt taatactaaa aggtatgtag tttctcctta tgagtaaaag 46740 tatttgtgta aaaatcctaa ttactttatt cttcctcaga attccatcat ccctggccct 46800 cccttcctat ttggagcctg ttcagtactg ctggctctgc ttgttgcctt gtttattccg 46860 gaacatacca atttaagctt aaggtccagc agttggagaa agcactgtgg cagtcacagc 46920 catcctcata atacacaagc gccaggagag gccaaagaac ctttactcca ggacacaaat 46980 gtgtgacgac tgaaatcagg aagatttttc tatcagcacc caggtcttag ttttcacctc 47040 tagttctgga tgtacattcc atttccatcc acagtgtact ttaagattgt cttaagaaat 47100 gtatctgcat gaactccgtg ggaactaaag gaagtgggaa cttagaacca gacagttttc 47160 caaagatgtt acaatttctt ttgaaaaacc ttttgtttat tagcaccaat ttcttgccac 47220 taagctattt gttttattat acatccttta attaaaaact atatatgtaa cttcttagat 47280 attagcaaat gtctctgcta ccatttcctt aaggtgttga gctttaactc tatgctgact 47340 cagtgagaca cagtaggtag tatggttgtg gacctatttg ttttaacatt gtaaaatttt 47400 gagtcagatt ttaatattgt aaaatcttgg gtcaaataat tcaaagcctt aatgcagatg 47460 cactaaaaca aagaaatggt aaatgaattg tttgcattta aaaaaaaaaa ctcttaagaa 47520 aactgtacta aatctgaatc atgttttgag cttgtttgca gtacttttaa acattattca 47580 ctactgtttt tgaagtgaga aagtatcagc catttagcat ttaagttggg gtatttagag 47640 cctgtaatct aaatgctggc tcaaatttat tccccagcta cttcttatac cactattctt 47700 ttaatgtttg cataatcata agcacctcaa cacttgaata cataatctaa aaattatata 47760 gtaaagctgg tagccttgaa aatgtcagtg tgatatctat tatgtagata aatatatata 47820 gtggcctttc aggactgtca cagtaacact ttatttacag agctaatgtt tgtcctaaat 47880 tttcaggacc ctagaggaga gctttataca attaccgatg tgaatttctc taaagtgtat 47940 atttttgtgt ccagttatat tatttaaaaa agtgttactt tgtaaaaatt gtatataaag 48000 aactgtatag tttacactgt tttcatcttg tgtgtggtta ttgcttaatg ctttttaaac 48060 ttggaacact cactatggtt aaataaggtc ttaaaagaaa tgtaaatatt ctgttaataa 48120 agttaaatat tttaatgatt ttttttttaa aaaagtcttg atctgtgagt tcctacaggg 48180 tctgttttgg ctttgttacc atatttttac agtaacaaat caaagcatta ttcattcttt 48240 ctttgcatcc atttcttttc cctcactaaa aaatgctctt caaggaagac aagaggaaag 48300 aaacaaaaat gcagtatgcg ttttgaagta cttgattttc aaagtgtaga tgctgcataa 48360 tagtttatta acaaagctca aagtttacag aaataacatt aaatgcaaca ctttttgatt 48420 attaacacat gtacaatttt acactgcaaa acaattgcaa ctaaaagttt aggaggtaaa 48480 gaattagcac acttggaagt ctgtatacat ttaatacaaa tttgcaagat atcagaatga 48540 tctctcctga aaattcaact ttccttgtgt atatatatgc aaagagatac ctatatttta 48600 aaaaagagag ctacctgtat gtcatgcatc ccatccaaaa catgtcacaa tattttatat 48660 atattacata gtatttacaa caaagcccct tccatagtat ttacaataaa gctccttcca 48720 aaatcaccaa gaggcttctt tccagaatag caagtgcttt caagttactg taccaagtat 48780 tctcactaat acagtagtgt atctaaatgg gggaggtggg ggagtatcaa ctgcctaata 48840 ttagttactg aattttcaga ttagatccct agtttaaaga cacttgcaat tgccaatagt 48900 ttcaagatac tggcttcgta aaaaaaaaaa aaaaattagc aaagattctt tcttaccatg 48960 ggactcagta atgttattac atcaatatct tgaaatgttt cttcatctgt gtaataaagt 49020 agttgaaaat aattttaaat tatcacatga gcattagtac tttataaaaa ttgatctaga 49080 agactcttta gagaatgcta ccaggtattg tttgtcaata agaaaacaaa atgaggccct 49140 atgattctag gtgaatttta aaaaaatttt tccccaagga acaattctta aaactttcag 49200 ttacagggaa gagaagaaaa ttgtaccttc taacagttat ttgttttgcc tagtttatta 49260 acattgaaat caacccaagt tctgatacta taaaaataaa tgaatattca tgatatagta 49320 gatttagtag tagtaccaag ttttaaaagt ccactgatac agatttaact gcataataaa 49380 actacaaatt gagaaaaact agatgagtat aaacatttag gaagcactga agttttaaaa 49440 acttgtcagc tcaaatgaca aaagcactta atctggtcac ataaaaactg tccaaattat 49500 tttaagtata tcaaatttat ttgattcatc actagcaaat ttaaatgctt caaggaaaat 49560 acgctatgaa ggcttaattc atttcagtta tttaaggtaa atttaggact tttcccttta 49620 aatgtcatca ataatatatc tcaagaggta atcattttcc ctcatgattt tcaggtgttg 49680 aatttaaaag ttttaccaat gaaagtgata aaatgaatta gtattctttg gttgcatact 49740 atgaggttat gagcaggttt tagtttaccc agatttttaa tatgatcaga atctccctca 49800 taaggatcaa ctctttcctt agaactgaat ttctaaagaa tagcttaata aaattcatat 49860 tattctataa aacgccatgt tcacaaacca aaaaatgtca tttactcaca atctttacca 49920 atccccaagt acaaatttgt tcctatatta taaactgcca taaaagcagt acttaaagta 49980 tcca 49984 6 490 PRT MUS MUSCULUS 6 Met Thr Gln Gly Lys Lys Lys Lys Arg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys Lys Ile Ile Ile Lys Asp Gly Gly Thr Pro Gln Gly Ile 20 25 30 Gly Ser Pro Ser Val Tyr His Ala Val Ile Val Ile Phe Leu Glu Phe 35 40 45 Phe Ala Trp Gly Leu Leu Thr Ala Pro Thr Leu Val Val Leu His Glu 50 55 60 Thr Phe Pro Lys His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val 65 70 75 80 Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser 85 90 95 Asp Val Trp Gly Arg Lys Ser Phe Leu Leu Leu Thr Val Phe Phe Thr 100 105 110 Cys Ala Pro Ile Pro Leu Met Lys Ile Ser Pro Trp Trp Tyr Phe Ala 115 120 125 Val Ile Ser Val Ser Gly Val Phe Ala Val Thr Phe Ser Val Val Phe 130 135 140 Ala Tyr Val Ala Asp Ile Thr Gln Glu His Glu Arg Ser Met Ala Tyr 145 150 155 160 Gly Leu Val Ser Ala Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala 165 170 175 Ile Gly Ala Tyr Leu Gly Gln Met Tyr Gly Asp Ser Leu Val Val Val 180 185 190 Leu Ala Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala 195 200 205 Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala 210 215

220 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val 225 230 235 240 Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe Leu Ser 245 250 255 Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr Leu Lys 260 265 270 Gln Ile Met Lys Phe Ser Pro Glu Ser Val Ala Ala Phe Ile Ala Val 275 280 285 Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile Val Leu Ser Leu Leu 290 295 300 Met Arg Ser Ile Gly Asn Lys Asn Thr Ile Leu Leu Gly Leu Gly Phe 305 310 315 320 Gln Ile Leu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Glu Pro Trp Met 325 330 335 Met Trp Ala Ala Gly Ala Val Ala Ala Met Ser Ser Ile Thr Phe Pro 340 345 350 Ala Val Ser Ala Leu Val Ser Arg Thr Ala Asp Ala Asp Gln Gln Gly 355 360 365 Val Val Gln Gly Met Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu 370 375 380 Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 385 390 395 400 Lys Glu Leu Pro Ile Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro 405 410 415 Gln His His Phe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu 420 425 430 Phe Gly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile 435 440 445 Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His 450 455 460 Cys Gly Ser His Ser His Pro His Ser Thr Gln Ala Pro Gly Glu Ala 465 470 475 480 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val 485 490 7 490 PRT MUS MUSCULUS 7 Met Thr Gln Gly Lys Lys Lys Lys Arg Ala Ala Asn Arg Ser Ile Met 1 5 10 15 Leu Ala Lys Lys Ile Ile Ile Lys Asp Gly Gly Thr Pro Gln Gly Ile 20 25 30 Gly Ser Pro Ser Val Tyr His Ala Val Ile Val Ile Phe Leu Glu Phe 35 40 45 Phe Ala Trp Gly Leu Leu Thr Ala Pro Thr Leu Val Val Leu His Glu 50 55 60 Thr Phe Pro Lys His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val 65 70 75 80 Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser 85 90 95 Asp Val Trp Gly Arg Lys Ser Phe Leu Leu Leu Thr Val Phe Phe Thr 100 105 110 Cys Ala Pro Ile Pro Leu Met Lys Ile Ser Pro Trp Trp Tyr Phe Ala 115 120 125 Val Ile Ser Val Ser Gly Val Phe Ala Val Thr Phe Ser Val Val Phe 130 135 140 Ala Tyr Val Ala Asp Ile Thr Gln Glu His Glu Arg Ser Met Ala Tyr 145 150 155 160 Gly Leu Val Ser Ala Thr Phe Ala Ala Ser Leu Val Thr Ser Pro Ala 165 170 175 Ile Gly Ala Tyr Leu Gly Gln Met Tyr Gly Asp Ser Leu Val Val Val 180 185 190 Leu Ala Thr Ala Ile Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala 195 200 205 Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Ala Ser Trp Gly Ala 210 215 220 Pro Ile Ser Trp Glu Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val 225 230 235 240 Gly Gln Asp Ser Ile Val Leu Leu Ile Cys Ile Thr Val Phe Leu Ser 245 250 255 Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr Leu Lys 260 265 270 Gln Ile Met Lys Phe Ser Pro Glu Ser Val Ala Ala Phe Ile Ala Val 275 280 285 Leu Gly Ile Leu Ser Ile Ile Ala Gln Thr Ile Val Leu Ser Leu Leu 290 295 300 Met Arg Ser Ile Gly Asn Lys Asn Thr Ile Leu Leu Gly Leu Gly Phe 305 310 315 320 Gln Ile Leu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Glu Pro Trp Met 325 330 335 Met Trp Ala Ala Gly Ala Val Ala Ala Met Ser Ser Ile Thr Phe Pro 340 345 350 Ala Val Ser Ala Leu Val Ser Arg Thr Ala Asp Ala Asp Gln Gln Gly 355 360 365 Val Val Gln Gly Met Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu 370 375 380 Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Ile Phe His Val Glu Leu 385 390 395 400 Lys Glu Leu Pro Ile Thr Gly Thr Asp Leu Gly Thr Asn Thr Ser Pro 405 410 415 Gln His His Phe Glu Gln Asn Ser Ile Ile Pro Gly Pro Pro Phe Leu 420 425 430 Phe Gly Ala Cys Ser Val Leu Leu Ala Leu Leu Val Ala Leu Phe Ile 435 440 445 Pro Glu His Thr Asn Leu Ser Leu Arg Ser Ser Ser Trp Arg Lys His 450 455 460 Cys Gly Ser His Ser His Pro His Ser Thr Gln Ala Pro Gly Glu Ala 465 470 475 480 Lys Glu Pro Leu Leu Gln Asp Thr Asn Val 485 490 8 426 DNA Homo sapiens 8 agtcatactg tattttttac ttgtattttt gttgttttgt gggatttaaa aaatattttt 60 attctgagga tagttgaatc cacaggatac tgagggccag ctgtattcac aacccaaatc 120 acatabaaag cgacaagttc atacacaata ggcctattag aacaggactg ttctctcttg 180 tttatcattg cagcctttct agcacaaagc ctgggacatt ctggacattt agtatgtgtt 240 aaatttctct tactacatta tttccaacag tatttactgc aatctgcaat taccttcctt 300 ttgttttgta actgtgtccc ccactagaat gtaagctctg tgcagatagt gtctcattta 360 ttgatgtatc cctggcatct aataaaacac tgacaacaca agcacccagt aaatattttt 420 tgaatg 426 9 413 DNA Homo sapiens 9 agtcatactg tattttttac ttgtattttt gttgttttgt gggatttaaa aaatattttt 60 attctgagga tagttgaatc cacaggatac tgagggccag ctgtattcac aavccaaatc 120 acatacaaag cgacaagttc atacacaata ggcctattag aacaggactg ttctctcttg 180 tttatcattg cagcctttct agcacaaagc ctgggacatt ctggacattt agtatgtgtt 240 aaatttctct tactacatta tttccaacag tatttactgc aatctgcaat taccttcctt 300 ttgttttgta actgtgtccc ccactagaat gtaagctctg tgcagatagt gtctcattta 360 ttgatgtatc cctggcatct aataaaacac tgacaacaca agcacccagt aaa 413 10 601 DNA Homo sapiens 10 caggagtggc taagctaggt ggtcctacct ctgggtctct catgaagttg tagtcagcca 60 aaggcttgac caaggttgga ggatctactt ccaaagtgac tcactccgtg gcatttggta 120 ggaggctaca aacagttcct ggacaactgg atctctccat aggctgcttg agtgtcctga 180 aaacacggaa gcaggcttcc ccaggctcca agccccaaaa tgaatgaaaa agagacccgc 240 aaaggaagat gcagtgcctt ttatgaccta gcctctgaag tcaatactgt cacttctgtt 300 ytgatctatc aagagtcact aagcctagtc tacactcaag gggaggggaa ttagagtcca 360 cctcttccag ggaggaatat cattgaatct gtgaacatat cttagaacta ccatacctag 420 tttcagtact tttaaacatt cgccattttg ctttgtccct ctcttttccc cacctacata 480 tacatacaca tacatgttac tccctaacca tctgagagta gggagcatgc ggtgtatccc 540 tatccctcgt gtttttctct taaggaaaag gatattctat tatacaacac ggtagttatc 600 a 601 11 601 DNA Homo sapiens 11 tcttaagtta cccattactt acggaaaatg attttttact gttcccttcg gttcctgtct 60 tggttagaac acagctggag attgtgttaa tagcttagga cgtctgtttc cgtgagcagg 120 taacaacttt ttgaaacaaa ttccctcatc tgctgaagaa gggggacaaa aacggcccct 180 atcgcccaga accgttgcga ggatttagct agctggtgac gccggagcac gaagttgtac 240 aggtagccag cagcacccac gcgagcccgc ggttaccctg gccgcgcggc tactgtagag 300 ygggctggcg gcgagcgggc ggggcggtat cacgcgggag gggcggggcc cgctcgtcgg 360 ctgatcgcac gattgtgacg cgccgccgga ggcaggccgg gccctcaaga tggcggcggg 420 cgcccagagc ggctcggccc ggcagtagtg gtgggacggc actagctgct ggggcctgcc 480 gccccgggag tggctgcagc agcgccagga atcgaggatg gtaaaatgac ccaggggaag 540 aagaagaaac gggccgcgaa ccgcagtatc atgctggcca agaagatcat cattaaggac 600 g 601 12 601 DNA Homo sapiens 12 ttttattacc tttctttggg gttttgtttg atttgcttta cagcagatgc tttctttcca 60 aatcctgtga gttttggaaa agatcgtttt taaactttct tgtcctatta ttaaggttgt 120 aattaattct tagcctgctt tgggacacaa aataaaatgt ttgcaccagc aataggtttc 180 acatagaaca aatgaagact tttcttgagg gctgtgaaca tgggggctat tatcatttct 240 catctttata cacttaatat ttcattctct attctaagag cactgggcac tcctttagaa 300 waggggcttt gttttgtatg tttggatccc acagggccta gtatgtgaat tttaaagtga 360 taaaaacact tctattttgt actagcacat tcctagatga atttttattg taattttgtt 420 tattcttata cgtaatcaga ggatatattt caataaatat caggggaata ttttgcatta 480 tttgtatttt aatccatccc agctttaaat ttaaaaagta taactattgc agtcatagaa 540 atgattgtaa aatggtagtt gcttatctac ctctctactt acaatagttc agactactat 600 t 601 13 601 DNA Homo sapiens 13 taatccatcc cagctttaaa tttaaaaagt ataactattg cagtcataga aatgattgta 60 aaatggtagt tgcttatcta cctctctact tacaatagtt cagactacta ttatgaactt 120 tttttgtttg tttgtttgag atggagtctc actctgttgc ccaggctgga ggagtgcagt 180 ggcaggatct cggctcactg taaccaccgc ctcctgggtt caagtgattc tcctgcctca 240 gcctcccgag tagctgggac tacaggcacg tgccaccatg cctggctaat tttttatatt 300 ktcagtagag acaaagtttc accatattgg tcaggctggt cttgaactcc tgacctcatg 360 attcacccac cttggcctcc caaagtgcag ggattacagg tgtgagccac cgtgcccaga 420 ctgaacattt tttaagaaag gggaaaaaat tgccatttga tactctgttg ttgtgtgttt 480 tttaattcat cgtatcatag aatatttcag tgctattgct gttgacctca gagtttcaga 540 gtttttataa agttccgcca atgggtagat tcattcagtg agatgtctga ggctctatgg 600 t 601 14 601 DNA Homo sapiens 14 caagtgattc tcctgcctca gcctcccgag tagctgggac tacaggcacg tgccaccatg 60 cctggctaat tttttatatt ttcagtagag acaaagtttc accatattgg tcaggctggt 120 cttgaactcc tgacctcatg attcacccac cttggcctcc caaagtgcag ggattacagg 180 tgtgagccac cgtgcccaga ctgaacattt tttaagaaag gggaaaaaat tgccatttga 240 tactctgttg ttgtgtgttt tttaattcat cgtatcatag aatatttcag tgctattgct 300 bttgacctca gagtttcaga gtttttataa agttccgcca atgggtagat tcattcagtg 360 agatgtctga ggctctatgg tcggtacatg acagtcgtga acagtatttc acatacctgg 420 tcaatggtac tgatttgatc ccccttctga tttcttcttt tcaacaatgt taataaaatt 480 ctttcccgtt gtcctgctaa tgacatatat gtaagcctat ttggccagtt taaatattta 540 taaacaaaac tagtaagagt tgttaatgat ttttctgaaa attagagcag attagagcag 600 a 601 15 601 DNA Homo sapiens 15 cttatctttt aaaatgctca tcttaaaata tgatctttat tgttttggcc atacaattgt 60 ggaactacat ctctgacagt ggaaaatgta tagttctttc agaagtttgt ggtaaaatga 120 ctttaaagat ttgatagaaa gtaaggcata tctgaattgc atggtcggaa gtacctgaaa 180 aaagtaaaat tgatatatca tttgaaaatg aaatgcatat ccctggataa gcagagcacc 240 agattttttt tttcttggca tccctgattt taattaaata ggagtcagca accgtttcaa 300 ragcaggacc caagctctga ccctttgcac tcttcacctg caaggatggc tgaagtagtg 360 gcaggaaagc tctctgggat gtagggcctt tgtagaccca gagagctgtt aaataacctt 420 tggttgctag catgcaagca ataagaaggg cctgtggtgc ttttcttttt ctttcttttt 480 ttttttcttt tgagacagag ttttgctctt gttgctcagg ctggggtgca atggcgtgat 540 cttggctcac agcaacctct gcctccctgg ttcaaggaat tctcctacct tagcctcctg 600 a 601 16 601 DNA Homo sapiens variation (301)...(301) A may be either present or absent. 16 taggacttct aaatttttta attactatgg gtacaaagta gatacagata tttatcaggt 60 acatctgata ttttgataca agcatatgtt gatacaggta tacagtgtat aataaatcag 120 ggatactggg gtatccatta cctcaaactt ttatcatttc tttgtgttag gaacatgcca 180 attccacttt tattttattt tattttttat tttttgagac agagtctcgc tctgtcgccc 240 aggcgacata catagtacag tagtgtactc cagcctgggt gacggggaga ctctgtctca 300 aaataaataa ataaataaat aaataaatct gttcagacta atgtcctaga gtgtattccc 360 aatgttttct tctagtcgtt tgtggtttca ggttttagat ttaagtcttt aatccatttt 420 gatttgattg ttgtacatgg caagaggtag gggtataatt ttattcttct gtatatggat 480 atccactttt cctagcacca tttaggagac tatccttttc ccaatgtata cttcggtgcc 540 gttgtcaaaa atgagttgac tgtaaatgca tggatttatt tctgggttct ctattgtgct 600 c 601 17 601 DNA Homo sapiens variation (301)...(301) T may be either present or absent 17 tgggtacaaa gtagatacag atatttatca ggtacatctg atattttgat acaagcatat 60 gttgatacag gtatacagtg tataataaat cagggatact ggggtatcca ttacctcaaa 120 cttttatcat ttctttgtgt taggaacatg ccaattccac ttttatttta ttttattttt 180 tattttttga gacagagtct cgctctgtcg cccaggcgac atacatagta cagtagtgta 240 ctccagcctg ggtgacgggg agactctgtc tcaaaataaa taaataaata aataaataaa 300 tctgttcaga ctaatgtcct agagtgtatt cccaatgttt tcttctagtc gtttgtggtt 360 tcaggtttta gatttaagtc tttaatccat tttgatttga ttgttgtaca tggcaagagg 420 taggggtata attttattct tctgtatatg gatatccact tttcctagca ccatttagga 480 gactatcctt ttcccaatgt atacttcggt gccgttgtca aaaatgagtt gactgtaaat 540 gcatggattt atttctgggt tctctattgt gctctattgt ctatgtatct gtttttatac 600 c 601 18 601 DNA Homo sapiens 18 tttgttttta atttttttga gacggagttt tgctcttgtt gcccaggctg gaatgcaatg 60 gcgcaatctt ggctcaccgc aacttccgcc tcccgcgttc aagcgattct cctgcctcag 120 cttcctgagt agctgggatt acaggcatgc gccaccacgc ctggctaatt ttgtattttt 180 agtggagacg gggtttcttc atgttggtca ggctggtctt gaactcctga cctcaggtga 240 tccacccgct ttggcctccc aaagtgctgg aattacaggt gagagccact gcgcccggcc 300 batgaatgat cttttttaag acctccttcc tgaaggaggt ttgctagtat tttgttgagg 360 atttttgcat caatgttcat cagagatatt gtcccatagt ttattttgtt tttctccatg 420 ctagttttag gtaatttttc tcttaaataa acaaagcatt ttcctcctaa agtgcaagca 480 tgcttattag aaaagatatg gaaaattcag aatagcatag taaacaatgt gatatcactt 540 aaaatcatta cctaatataa attttattta cattgaggtc agtatttatt gtttttcaga 600 g 601 19 601 DNA Homo sapiens 19 ccgcaacttc cgcctcccgc gttcaagcga ttctcctgcc tcagcttcct gagtagctgg 60 gattacaggc atgcgccacc acgcctggct aattttgtat ttttagtgga gacggggttt 120 cttcatgttg gtcaggctgg tcttgaactc ctgacctcag gtgatccacc cgctttggcc 180 tcccaaagtg ctggaattac aggtgagagc cactgcgccc ggccgatgaa tgatcttttt 240 taagacctcc ttcctgaagg aggtttgcta gtattttgtt gaggattttt gcatcaatgt 300 ycatcagaga tattgtccca tagtttattt tgtttttctc catgctagtt ttaggtaatt 360 tttctcttaa ataaacaaag cattttcctc ctaaagtgca agcatgctta ttagaaaaga 420 tatggaaaat tcagaatagc atagtaaaca atgtgatatc acttaaaatc attacctaat 480 ataaatttta tttacattga ggtcagtatt tattgttttt cagagttgaa attaccctac 540 ctatacatgt tatatcctac tttgattttt aaaaaaatta gcatgcttta agccctgaga 600 a 601 20 601 DNA Homo sapiens 20 gttcttgatt ctcccttccg ctatttatca agctcctccc aatttcaaat cctgaatcct 60 taatccgttc cctcccctcc aacattcata ctgtgccact gttttatgcc ctcatttctt 120 gtttgagctg attcagatag cttcctttta gatgcgcttt gcttctccat tttatccttt 180 aggaaatcac cagagtgata atactgcagt gagtcttaag acatctctgg cagcggtata 240 aacttaattt tgtattttct ttctcatgta tatcaaattc caaatctctt acatactttc 300 sctggggatt gttctgcttt tgagccatgt tgatatcgtg tttatatttt tgccacttgc 360 ttcatttatg gttttttttt ttttttttgg ttacatcttt gccagaataa tcttaaaact 420 ttcatctgat tgtgtcagtc ttaatatctt ttagtggctc cccatggcct tcagaattaa 480 atatagactc cttagcatgg aagctggtct ttgagtacct gtagcttgtc tttcaataca 540 cccaacgtgc agcccatgca ctggttgtac tgaactcgat atatgagacc cataatgccg 600 c 601 21 601 DNA Homo sapiens 21 acaggctgag ccatcttttc caccctatac ctccgcctgt ctaactctgt tgtgtccttt 60 cagccttcct cctggaagtc tgatatttcc cacctcccaa gctcccttgg actctgtatg 120 ttccaactgc atactgtgct tatgctaatg aatttcgttg ttgccttgtc tgtccctctg 180 actttgaaga cagaggcagt gagtacagat gtttgacaca gtgcccagta catatatgat 240 cttaatattt gttgactatt aacatcgttg ttattgttaa taattataga atgtactgtt 300 wacttttttt aactttttaa aaaatcttgt tttttatagc ctcaaggaat aggttctcct 360 agtgtctatc atgcagttat cgtcatcttt ttggagtttt ttgcttgggg actattgaca 420 gcacccacct tggtggtaag taatctttta aattatttaa cactgactcc aaaatctctt 480 cttcttcagt tttggaggaa aatgtgggcc ttttcccttt gcacggttaa ttctcccacc 540 agtattgttc agtattcacc agtattttac tggttgtctt ttccaactgt taactctccc 600 t 601 22 601 DNA Homo sapiens 22 gaggaaaatg tgggcctttt ccctttgcac ggttaattct cccaccagta ttgttcagta 60 ttcaccagta ttttactggt tgtcttttcc aactgttaac tctcccttac ctttttttgg 120 gaggggggtg gcgtggaggt gtttgaattt ggacttgtca ctgggcatgt tcaagcagag 180 gctctgtaac tactctgagt aaaatggaag agattcttaa accgacaggt ttagaaaaga 240 tgatgtctgt gacctgcatg actcggcata attactttga ggttcattta tgcagctgta 300 vtttccaaaa acaggtttct gttcatttgg gctaagtacc tagaagggct attctttaat 360 agatctaagc tgattttacc caaattctcc caggtttgaa actttagaaa agacctccct 420 gcccgaccaa acaactcaga agatagccag ttttcttata ttggtgtaga taaggggaat 480 ggaaggaggg aaggactatc tatggtaaat atctatacca tcttgaaagg agtaattatg 540 ataaatgtac agtttaccaa atcctagagg aatagagttt taaagtaata tactatgttt 600 t 601 23 601 DNA Homo Sapiens 23 aaaaaaaaac agccgggcgc ggtggctcac acctgtaatc ccagtacttt gggaggccaa 60 ggcgggtgga tcacgaggtc aagagattga gaccatcctg accaacatgg tgaaaccctg 120 tctctactaa aaatacaaaa attagctgtg cgtggtggta cgcacctgta atcccagcta 180 cttgggaggc tgaggcagga gaatctcttg aacccgggaa gtggaggttg cagtgagccg 240 agactgcacc actaccctcc agcctggata cagggtgaga ctctgtctca aaaataaaaa 300 rtcattttga atatatagag catgttcatg agtattgcta taaaaaaata tcagagggtt 360 tttttttttt ttttagttta

ctgatttcag atagaaatct ttaaaaaatt aatttacaca 420 tttcctggct tcataatcca agtacaacga tttggaactt cctcagatga tgcaagttga 480 ttatgacatt cataacttca ttgaattgta ataacctgtt tttgtcaagg gttactgaag 540 tgctgtaata actttttggg ctcatgactt tacattagct ttcctaatgc gccagcgtgc 600 t 601 24 601 DNA Homo sapiens 24 cttgactcct ccagtgctca gtcttttggg gaatgcaggt agtaacttgt ttgtacccat 60 gttttagata gttgaggttg tcaggcagcc caaccactag ctaagtaggg tgatcaaaat 120 gtggatgagc tgttagcaag ctatgaaaaa aagcattttg tgatgtttcc ataatttgtt 180 atcagtattt caagtgtgta tagctatttt taaaatttgc ttcttgttta aattttttta 240 ggtatgttat ctttcgtgtt attttggtac atttttttcc tagttggaca aagggaggct 300 htctttttta agaacaagga aggagtcccc ttaattagaa aggcttgttt attcattttt 360 catagactaa tgtgcttaat atattccttt tttttttttt tttttttttt gagacggagt 420 ctcgctctgt ctgtccccag gctggagtgc agaggcacga tcttggctca ctgcatcccc 480 cacctcccag gttcaagtga ttttcctgcc tcagcctccc aagtagctgg gactacaggc 540 acatgccacc atgcccagct aatttttgta cttttagtag agatggggtt tcaccatgtt 600 g 601 25 601 DNA Homo sapiens variation (301)...(301) T may be either present or absent 25 accactagct aagtagggtg atcaaaatgt ggatgagctg ttagcaagct atgaaaaaaa 60 gcattttgtg atgtttccat aatttgttat cagtatttca agtgtgtata gctattttta 120 aaatttgctt cttgtttaaa tttttttagg tatgttatct ttcgtgttat tttggtacat 180 ttttttccta gttggacaaa gggaggctat cttttttaag aacaaggaag gagtcccctt 240 aattagaaag gcttgtttat tcatttttca tagactaatg tgcttaatat attccttttt 300 tttttttttt ttttttttga gacggagtct cgctctgtct gtccccaggc tggagtgcag 360 aggcacgatc ttggctcact gcatccccca cctcccaggt tcaagtgatt ttcctgcctc 420 agcctcccaa gtagctggga ctacaggcac atgccaccat gcccagctaa tttttgtact 480 tttagtagag atggggtttc accatgttga ccagaatggt ctcgatctct taacctcgtg 540 atccgcccgc cttggcctcc caaagtgctg ggattacagg tgtgagccac tgtgcctggc 600 c 601 26 601 DNA Homo Sapiens misc_feature (1)...(601) n = A,T,C or G 26 ttcaaagata ttctttgaag tattttttta atcagataac cagttttaga catattaatt 60 ttgaatgtct ggtttgggat ttatgatagc cttaatttct taatttttaa aactaatgtg 120 acattttaag accaaaaaaa ctgtgtgttg caattatctt tcacttttaa gccctcatag 180 aacagtcaaa aaacaaaagc tgtgttttgt ggaagatctg cccaggggaa gatggtgagc 240 ctctaccaac aaggggattt agctaaaaag aaggattttg tactgacaaa tatttttaaa 300 nattgaggtc taacactttt gagaggttat gaatatatgg ttggtcatag tagatagttc 360 agtcagaatc agtgattatt gcttgattat gtaacatatt agctaagtga tgagaataac 420 agtaggtata aggatctgta atgccaagga gtggaattta ccggtttttt tttttctttc 480 cttttttttt tttttcattg agacggagtc ttaatctggc atccaggttg gagtgcagtg 540 gcgtgatctc ggctcactgc aacctccacc gccaaggttc aagagattct cctgcctcag 600 c 601 27 601 DNA Homo sapiens variation (301)...(301) T may be either present or absent 27 aaaagctgtg ttttgtggaa gatctgccca ggggaagatg gtgagcctct accaacaagg 60 ggatttagct aaaaagaagg attttgtact gacaaatatt tttaaagatt gaggtctaac 120 acttttgaga ggttatgaat atatggttgg tcatagtaga tagttcagtc agaatcagtg 180 attattgctt gattatgtaa catattagct aagtgatgag aataacagta ggtataagga 240 tctgtaatgc caaggagtgg aatttaccgg tttttttttt tctttccttt tttttttttt 300 tcattgagac ggagtcttaa tctggcatcc aggttggagt gcagtggcgt gatctcggct 360 cactgcaacc tccaccgcca aggttcaaga gattctcctg cctcagcctc cccagtagct 420 ggaattacag gtgcatgtca ccacgcccag ctaatttttt tttttattat tttttttgag 480 acagagtttc actctgtcgt ctaggctgga attcagtggc actatctcgg ctcactgcaa 540 ccttcgcctc ccaggttcaa gcagttctct gcctcagcct cccaagtatg tgggattaca 600 g 601 28 601 DNA Homo sapiens 28 cccacctaag cctcccaaag tgctgggatt acaggcgtga gccactgttc ctggccggct 60 ttaccctttt gacagaccta tggctctgga aataataggc cagtgtttga tggttcaagc 120 tcctagatac acagtccatg ttacggaaca ctcaaaatcc actagcatct cttctaccta 180 gatggtttcg tgtccttggc tacagaaaca gccccaaagc gtttaacatt ttaaggatta 240 tttactttca acatttttaa agttaaaaaa aagttaagat ccataaaatt ttttggaaaa 300 rtgttacatt ttctctgttc acctctaaag accagtgcta aaggatcctg acatcaaaaa 360 tctttacaac attcgaatta cttgttatat ttgtctgtta aaattttgtt agaaattgta 420 tggccccaaa ggagaaattg ctttggagaa aaaagttagg tagcagagga acagtttgga 480 agggttgggg gttggccaga taaagaaagg gaagaaacat tcaaaattga aaggatgccg 540 tgtataaaat atgaatattg gaaagcatag aatatttcag aaacagtgaa gcgaacagat 600 t 601 29 601 DNA Homo sapiens 29 aggaggctga ggcaggagaa ttgcttgaac ctgggaggcg gaggttgcag tgagccaaga 60 ttgcaccatc gcactccagc ctgggcgaca agagcccaac tccgtctcaa acaaacaaaa 120 aaaggaatag tgctgcagta aatgtaggag tacagctatc tcttcaatat actgatttcc 180 tttttttgga ggggtatata cctagtagtg agattgctgg atcatatggt agctccattt 240 ttaggttttt tgaggagcct tccaactgtt ttccttagtg attgtactaa tttacattcc 300 vaccaacagt gtatgagtgt tcccttttct ccacatcctt gcctatcttt tggataaaag 360 ctgtttttaa ctggggtgag atgatatttc actgtagttt tgatttgcat ttccccgatg 420 atcagtgatg gttgagcatt ttttcatata cctattggtc actttgagaa atgtctattc 480 agatcttttg cccgtttttt aaaaatcaga ttatgagatt cttttcttac agaattgttt 540 gagcccctta tacatttttg ttattaatcc cttgtcagat ggatagtttg cagatatttt 600 c 601 30 601 DNA Homo sapiens 30 gcacattttt gttaaatttc tcgctattgg caggagaaga ataactgaag aaaggggagc 60 aattctgatc cttctaaagg ttcttcttgc aacatgtcag aaagtatatt tagcataatg 120 tttcttctta aagggaagac cttccctacc ttccttatta cccacattcc cattctctgt 180 tgttattact gagcgatagc attggataat agaagcatta gtttctaagt caaacaggaa 240 ctcagttgcc tcatatgtaa agtgataata ttatctaatt cacagtgttg ggattaaaca 300 rgagtacata taggctgtaa aaatggtagc tgctgtttat ttttccagtt gcctggaatt 360 gccttttcat ttgatgcatt ccagcggttc tcttgctgcc cactgcaaaa aattgatacc 420 acatgatttg agaacaagcc ttggaaagga tagaataact tgttatacat tttcataggt 480 tgggattttt tttctttata gaatctttct agatctactt cgtggcaatt aaaaattact 540 tattaatttt cccaatctcc tatcctagat aatatatcca tctgaaagag aattataagt 600 c 601 31 601 DNA Homo sapiens variation (301)...(301) A may be either present or absent 31 agacaaatac cttgtgataa ataaggactg aatattgtgt tgggctgaat tagttttaaa 60 agggactgat ttctgattca aaggacgtta tagtgaagaa tcataagatt tttggggagg 120 aaacacctat agagagaaag ttagaaaaag aactaataat ttctggcctg ttcagtggct 180 cacacctgta atctcagcac tttgggaggt tgaggcaggc ggatcacttg agatcaggag 240 ttcacgacca gcctggccaa catggtgaaa ccttgtctct atttaaaaaa aaaaaaaaaa 300 aaaaagtgaa aagaaaaaga actaatgatt tcagttgtaa acttggaaca ttaaatgata 360 caaggctgat gatagccagg atatttaaaa aatagtctaa ttaagctata gtttacatac 420 cataaaattt atccttttta tgagtatagt tcagtgaatt ttagtaaatt tatactgtta 480 tgcaaacacc accataaccc aatttggggt tggtcggttg gttggttggt tcgttggttt 540 ggtttttttg acgtaattta ttttcccata gccaaagttt tgaaattaac aattttcaat 600 c 601 32 601 DNA Homo sapiens variation (301)...(301) A may be either present or absent 32 gttgtaaact tggaacatta aatgatacaa ggctgatgat agccaggata tttaaaaaat 60 agtctaatta agctatagtt tacataccat aaaatttatc ctttttatga gtatagttca 120 gtgaatttta gtaaatttat actgttatgc aaacaccacc ataacccaat ttggggttgg 180 tcggttggtt ggttggttcg ttggtttggt ttttttgacg taatttattt tcccatagcc 240 aaagttttga aattaacaat tttcaatctg gaggttctgt gtattaagcc atgttctggc 300 aaaaaacaaa acaaaacaaa acaaaacaaa acaaaacaaa aaacactgaa atcttctaga 360 aataatatgg atgcagaaaa aaggtgggga agtggccagg cacagtggca tgtgcctgta 420 ataccaccag tttgggaggc caaggcaggg ggattgcttg aggccaggag tttgaggctg 480 cagctatgat catgccacta cactccagtc tagggtacag agtgagaccc tgtctcttaa 540 aaaaaaaagt tggaggggcc aggtgcagtg gcttataatc ccagcacttt gggaggctga 600 g 601 33 601 DNA Homo Sapiens variation (301)...(301) C may be either present or absent 33 aacttggaac attaaatgat acaaggctga tgatagccag gatatttaaa aaatagtcta 60 attaagctat agtttacata ccataaaatt tatccttttt atgagtatag ttcagtgaat 120 tttagtaaat ttatactgtt atgcaaacac caccataacc caatttgggg ttggtcggtt 180 ggttggttgg ttcgttggtt tggttttttt gacgtaattt attttcccat agccaaagtt 240 ttgaaattaa caattttcaa tctggaggtt ctgtgtatta agccatgttc tggcaaaaaa 300 caaaacaaaa caaaacaaaa caaaacaaaa caaaaaacac tgaaatcttc tagaaataat 360 atggatgcag aaaaaaggtg gggaagtggc caggcacagt ggcatgtgcc tgtaatacca 420 ccagtttggg aggccaaggc agggggattg cttgaggcca ggagtttgag gctgcagcta 480 tgatcatgcc actacactcc agtctagggt acagagtgag accctgtctc ttaaaaaaaa 540 aagttggagg ggccaggtgc agtggcttat aatcccagca ctttgggagg ctgaggcagg 600 a 601 34 561 DNA Homo Sapiens 34 agcagaaaga aatggctacc aagtggagag aactgaggag aagggaaatg acatgaaaca 60 actgtactga cttgctcact gtgtcacaaa tgtgatctct gtaaatgccc tcaaatgtct 120 tcagtgaccc tcatagtgag aaccattttc cctttcccca cacttgtgcc agagccctgc 180 tgagatctgg gtccctctga aaccacacct agggctgcaa taacaaaata accactacat 240 ttgaaaatat atatttatat gtatgtgtgt gtgtgtatgt rtgtgtgtgt atatatatat 300 agtttgtttt ttgttgagac ggagtctcgc tctgtcaccc aggctggagt gcagaggtgt 360 gatcttggct tactgcaacc tccgcctcct gggttcaaac gattctgctg cctcagcctc 420 cccagtagct atgcccacca ccatgcccag ctaatttttg tatttttagt agagacgggg 480 tttcaccata ttggccagtc ttgtcttgaa ctcctgacct ttggtccgcc tgcctcggcc 540 tcccaaagtg ttgggattat a 561 35 385 DNA Homo Sapiens 35 agcagaaaga aatggctacc aagtggagag aactgaggag aagggaaatg acatgaaaca 60 actgtactga cttgctcact gtgtcacaaa tgtgatctct gtaaatgccc tcaaatgtct 120 tcagtgaccc tcatagtgag aaccattttc cctttcccca cacttgtgcc agagccctgc 180 tgagatctgg gtscctctga aaccacacct agggctgcaa taacaaaata accactacat 240 ttgaaaatat atatttatat gtatgtgtgt gtgtgtatgt atgtgtgtgt atatatatat 300 agtttgtttt ttgttgagac ggagtctcgc tctgtcaccc aggctggagt gcagaggtgt 360 gatcttggct tactgcaacc tccgc 385 36 361 DNA Homo Sapiens 36 agcagaaaga aatggctacc aagtggagag aactgaggag aagggaaatg acatgaaaca 60 actgtactga cttgctcact gtgtcacaaa tgtgatctct gtaaatgccc tcaaatgtct 120 tcagtgaccc tcatagtgag aaccattttc cctttcccca cacttgtgcc agagccctgc 180 wgagatctgg gtccctctga aaccacacct agggctgcaa taacaaaata accactacat 240 ttgaaaatat atatttatat gtatgtgtgt gtgtgtatgt atgtgtgtgt atatatatat 300 agtttgtttt ttgttgagac ggagtctcgc tctgtcaccc aggctggagt gcagaggtgt 360 g 361 37 601 DNA Homo Sapiens 37 agtagagacg gggtttcacc atattggcca gtcttgtctt gaactcctga cctttggtcc 60 gcctgcctcg gcctcccaaa gtgttgggat tataggcgtg agccatggcg cctggccccc 120 atgtgaatat attaaatacc atttaaaaaa ccaccacaac ccagttatag aacatttcca 180 tcagcccaaa atgttccctc agccctgttt gccatctgtc cccatgctcc acctgtgacc 240 ccaagcaacc aacaatttag cttctgtcac catggttttg ccttttctag aaacttcata 300 kaaattaaat aatacaaaac atcttttgtg tctaacttct ttcacttggc ataatctttt 360 gagattgatc catgttgata ctatagatca ataggttcta tttttgtctc ttttcctttt 420 tttttttttt gagacagggt cctgctctat cccccaggct ggagtgcagt ggcatgatca 480 tggctcactg aagccttggc ttcctgggct caagcgatcc ttctgcctca gcctccaaag 540 cagttgggac cacaggcatg atccaccatg cccagctaat ttttttcttt ttgagacagg 600 g 601 38 601 DNA Homo Sapiens 38 gcccttaacc aagcttgtcc aacccatggt ccacaggctg cacacatggc ccaacagaaa 60 ttcataaagt ttcttaaaac attatgcagt ttttttttct ttaagctcat cagctattgt 120 tagtgtattt tatgtgtggc ccaagaccgt tcttccagcg tggcccaggg aagccaaaag 180 attagacacc cctcccctaa ggaccagcat gactggcagt caaggagggg tgtttgtaca 240 gtgcccaggc tctcaaccct tcctcaacta aaagagttaa aaaatttaaa taggccgggc 300 rtggtggctc acgcctgtaa tcccagcact ttgggaggcc gaggcgggcg gatcacgagg 360 tcaggagatc gagaccatct tggctaacac gggggaaacc ccatctctac taaaaataca 420 aaaaattagc cgggcgaggt ggcgggcgcc tgtagtccca gctattcggg aggctgaggc 480 aggagaatgg cgtaaacccc gggggcggag cctgcagtga gccgagatcg cgccactgca 540 ctccagcctg ggcgacagag cgagactccg tctcaaaaaa aaaaaaaaaa aaaaaaaaaa 600 t 601 39 601 DNA Homo Sapiens 39 ctttaagctc atcagctatt gttagtgtat tttatgtgtg gcccaagacc gttcttccag 60 cgtggcccag ggaagccaaa agattagaca cccctcccct aaggaccagc atgactggca 120 gtcaaggagg ggtgtttgta cagtgcccag gctctcaacc cttcctcaac taaaagagtt 180 aaaaaattta aataggccgg gcatggtggc tcacgcctgt aatcccagca ctttgggagg 240 ccgaggcggg cggatcacga ggtcaggaga tcgagaccat cttggctaac acgggggaaa 300 vcccatctct actaaaaata caaaaaatta gccgggcgag gtggcgggcg cctgtagtcc 360 cagctattcg ggaggctgag gcaggagaat ggcgtaaacc ccgggggcgg agcctgcagt 420 gagccgagat cgcgccactg cactccagcc tgggcgacag agcgagactc cgtctcaaaa 480 aaaaaaaaaa aaaaaaaaaa aatttaaata gaggcagggt cttgctgtgt tgcccaggct 540 ggtcacaaac ttctggcttc acgcagtcct cccaccttgg cctccccaag tgctgagatt 600 a 601 40 601 DNA Homo Sapiens 40 ccaggcttgg tgtctcatac ctgtactccc agcccttcgg gaggctgagg tgggaggatc 60 gcttgagctc aggagttcga gactagccta ggcaacatag cgtgacttcc acctctataa 120 aaaataaaca aaattagctg ggcgtggtgg tgtgtgcctg tagccccatc aggagatctt 180 caggcaggaa gatctacttg agcctgagag gtcaagacta cagtgagccg tgatggcacc 240 actgcactcc agcctgggcg acagagcaag acccagttcc cccactctcg cccccacaag 300 raaaaaagat aaatggcaca ggtaggaaga gaaaagggag ggtgtgcaac agaaggcctg 360 acataaatca agattatgaa aggagttatg tggtgttgag gaaaaaagta gcctgactaa 420 tctctgtcta tccttaattt attgcaggtt tcagcaacat ttgctgcaag tttagtcacc 480 agtcctgcaa ttggagctta tcttggacga gtatatgggg acagcttggt ggtggtctta 540 gctacagcaa tagctttgct agatatttgt tttatccttg ttgctgtgcc agagtcgttg 600 c 601 41 601 DNA Homo Sapiens variation (301)...(301) R may be either present or absent 41 tactatttct agcttcaggt ttgtcctaaa aatcatcagt ctggaaaaac aatgcattta 60 aatattcatt cctagccatg agaaaagtgc tttttaactt tggaggaaaa tatactgtag 120 cctttatata aaaatggctt taaaaaaagt ttttgaggcc aggtgcggtg gttcatgtct 180 gtattcccag cactttgggt ggccaaggtc agggaccgct tgagcccagg agttcaagac 240 cagctcaagc aacttggcaa aaccccatct ctaccaaaaa aaaaaaaaaa aaaaaaaaaa 300 rgaaagaaag cccggtgtgg tggtgtgtgc ctgtagtccc agctactcag gaagctgaga 360 tgggaggatt gcttgatcct gggaggtcga gggtgcagtg agccacagtt gtcccatggt 420 actccagtat gggcaacaga atgagaccct gtctcaaaaa aaaagtgtaa ggaaaataca 480 cagttagtat gtgtagaact tgatgaatta tcaaagatta acccaacctt gcaatagatg 540 tgatcaacct gggcatgagt atctttttca tacattgcca acattagatt tgctaacatt 600 t 601 42 601 DNA Homo Sapiens 42 tcagccccat tacttcctgg gcaccggtac gtataagagt tcctaacact tgcactaagt 60 aagtgtttac atgagaacat caatatagtt ctacacattt cttttttctc agtgttttcc 120 tatccagccc tctctgtggg ggtgactccc acacttactc ttccagtcca gtccctgagc 180 tcctatatga cacattggct gggtatctca gccttaacat ggccaaaatt aaaatctggg 240 ttccatccct tgcccgccac ccctatgctc ctcatctcat tcagtggctt caccaccgcc 300 wggttttggg ggccagaagc cttagcgaca ttcatgaatc ctttctccct aaccttacat 360 tcagcccatc aaatgatact tcctatcacc tctctctcca aaatatatct tgaatcagag 420 cgtttctgat attctccatt agtaggaacc caatctgagc cgtggccata tcttccatct 480 ggtctctctg cttccatctt gcctcactat agttcattct gcacttggca gagtaatttt 540 tgtaaaatgg aaatttaatc gcatcttacc tataactcac tttcctttgc aaccagaata 600 a 601 43 601 DNA Homo Sapiens 43 attagttaga acttttgacc cttccatttt caactactga tttaagtggt cctcagtaga 60 aatgtactga ataggaagtt ttatctttca gttttctaac tctcagtctg gatctatgtc 120 agcagaggga cttttcatct gcttatgtga cctggactag tgatctcaga catattcagg 180 gcaattattg ctgaaaatca gccaaattgt agaaaagtgc caatagtcct tttatagtgt 240 agattgaaag aagtcacttt ttaaaacttt attctgataa atcttttttt ttttttttca 300 raccatagtc ttgagtttac ttatgaggtc aattggaaat aagaacacca ttttactggg 360 tctaggattt caaatattac agttggcatg gtatggcttt ggttcagaac cttggtaagt 420 ataaatattt taatgttaat atttttaatt ttggtgttag cccttgtgtt tttatttgct 480 tctcaactga ggggtagact gtaatctgtc tcatactatg ctttttatct ttcaaaatgt 540 gtctaatata agtctgccac ttgtatattt atatgttctc ctagaatggc ttgaggatta 600 a 601 44 601 DNA Homo Sapiens 44 taagtggtcc tcagtagaaa tgtactgaat aggaagtttt atctttcagt tttctaactc 60 tcagtctgga tctatgtcag cagagggact tttcatctgc ttatgtgacc tggactagtg 120 atctcagaca tattcagggc aattattgct gaaaatcagc caaattgtag aaaagtgcca 180 atagtccttt tatagtgtag attgaaagaa gtcacttttt aaaactttat tctgataaat 240 cttttttttt ttttttcaga ccatagtctt gagtttactt atgaggtcaa ttggaaataa 300 raacaccatt ttactgggtc taggatttca aatattacag ttggcatggt atggctttgg 360 ttcagaacct tggtaagtat aaatatttta atgttaatat ttttaatttt ggtgttagcc 420 cttgtgtttt tatttgcttc tcaactgagg ggtagactgt aatctgtctc atactatgct 480 ttttatcttt caaaatgtgt ctaatataag tctgccactt gtatatttat atgttctcct 540 agaatggctt gaggattaaa aaggtgacct tttatagcta aatgacaggc tgaatttttg 600 a 601 45 601 DNA Homo Sapiens 45 tctggatcta tgtcagcaga gggacttttc atctgcttat gtgacctgga ctagtgatct 60 cagacatatt cagggcaatt attgctgaaa atcagccaaa ttgtagaaaa gtgccaatag 120 tccttttata gtgtagattg aaagaagtca ctttttaaaa ctttattctg ataaatcttt 180 tttttttttt ttcagaccat agtcttgagt ttacttatga ggtcaattgg aaataagaac 240 accattttac tgggtctagg atttcaaata ttacagttgg catggtatgg ctttggttca 300 raaccttggt aagtataaat attttaatgt taatattttt aattttggtg ttagcccttg 360 tgtttttatt tgcttctcaa ctgaggggta gactgtaatc tgtctcatac tatgcttttt 420 atctttcaaa atgtgtctaa tataagtctg ccacttgtat atttatatgt tctcctagaa 480 tggcttgagg attaaaaagg tgacctttta tagctaaatg acaggctgaa tttttgaatg 540 agattataca gcttttgaat ctttaaggag

catttaatct aaatcagtcc gttactagga 600 a 601 46 601 DNA Homo Sapiens 46 tcatgtgttt ggaaatattt gatgatgttt cgtcatctat attatacatt atcctcaata 60 taacctctca attgcctgta gaaataatac ccagcacatt ttacagtttg gaacatgata 120 tccctatttt acattacacc ctcacagcac tcttgtgaat taagttgctg tctttgtata 180 cagggtcaga ttgttaagcg acttgcccat agtcacctag taagcaagtt cagttctcct 240 tttctctaca gccatttcac agtaagaatt acttaattat gtagtttgac tttcaggtac 300 rgtggagaag aatttactgt ttttgttttg ctgctctcct tataggatga tgtgggctgc 360 tggggcagta gcagccatgt ctagcatcac ctttcctgct gtcagtgcac ttgtttcacg 420 aactgctgat gctgatcaac agggtgagtt gataggaact agcgataatt atttaaaagt 480 acagaatgtt ctaatcctgt gttctgtctc ctatgtactg aaacataagt atatcttcag 540 ggtagagact tttaaaattg cttttgatat aaacaggaaa agcagattct agggtattta 600 t 601 47 601 DNA Homo Sapiens 47 cttaggtaga tacatattcc cttttctctc acttagaata tgtggcttat ctgttctgtt 60 catagaaaat ttactgatga ggctgggcat ggtggctcac acctgtaatc ccaacacttt 120 gagaggccga ggcaggcaga ttgcttgagt tcaggagttg gagaccagcc tgggcaacat 180 ggggaaaccc catcactaaa atgcaaaaat tagctgggca gggtggcgca tgcttgtagt 240 cccagttact tgggaggctg aggttggagg atcacttgag tctcagaggc ggaggttgca 300 stgagccaaa atcaggccag tgaactccaa cctgggcgac acagtgagag accttgagag 360 acctgtctca aaaaaattta ctgatgaatg ttggccacag aatattaact aagcattaag 420 tttttgctgt gttgtgctag acaccttgtg ggatatattc atagaccttt tatgaatgtt 480 gtcccagcca cttgggaagc tgaggcagga ggattacttg agctcaagag tttgaagcta 540 gcttaggcaa cacagcaaga ctcccatctc taataaaaaa aaaaaaagaa atcatatagt 600 t 601 48 601 DNA Homo sapiens 48 agcttttatg tgagattcta tttgagattg gataaaatgc ttaaaaacca gagtttaagg 60 gactgtgttc ttctacatac caccttgtga aaattggctg tgcatatttt ttttccactt 120 gagaatgaaa aattttaagt acctagtttg tcaatggcat atgtaacaaa ccatttctct 180 ttactactag tctcttttga aacttttcta atatcacagt tgtgtatgtt aactaacttt 240 tcatacaaaa ggcaggctta tggtaaataa cctttgttca ctgtttgcta tatttccctc 300 dtttcaactg aaaattaatg ccaaacaatg ctgatcattt tggccaatat tagcagctca 360 tcagtttcct ggcttattta gcagagttct gtacttatct tccaacccac taagactact 420 tttaaataaa agaatgtttg tgggctatac acaaaactgg cagtgcaagc ttctgctaag 480 aatgaatgta attttgggct aaaacaaagg tctctttttc ttggtaacct gtgtttttct 540 cacttccagt cacttgaaat tataattacc ttcttgaaac aaagaaatgg taatttattt 600 t 601 49 601 DNA Homo sapiens 49 cacagccatc ctcataatac acaagcgcca ggagaggcca aagaaccttt actccaggac 60 acaaatgtgt gacgactgaa atcaggaaga tttttctatc agcacccagg tcttagtttt 120 cacctctagt tctggatgta cattccattt ccatccacag tgtactttaa gattgtctta 180 agaaatgtat ctgcatgaac tccgtgggaa ctaaaggaag tgggaactta gaaccagaca 240 gttttccaaa gatgttacaa tttcttttga aaaacctttt gtttattagc accaatttct 300 dgccactaag ctatttgttt tattatacat cctttaatta aaaactatat atgtaacttc 360 ttagatatta gcaaatgtct ctgctaccat ttccttaagg tgttgagctt taactctatg 420 ctgactcagt gagacacagt aggtagtatg gttgtggacc tatttgtttt aacattgtaa 480 aattttgagt cagattttaa tattgtaaaa tcttgggtca aataattcaa agccttaatg 540 cagatgcact aaaacaaaga aatggtaaat gaattgtttg catttaaaaa aaaaaactct 600 t 601

Claims

That which is claimed is:

1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4; (b) an amino acid sequence of an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (c) an amino acid sequence of an ortholog of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; and (d) a fragment of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said fragment comprises at least 10 contiguous amino acids.

2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4; (b) an amino acid sequence of an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (c) an amino acid sequence of an ortholog of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; and (d) a fragment of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said fragment comprises at least 10 contiguous amino acids.

3. An isolated antibody that selectively binds to a peptide of claim 2.

4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).

5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).

6. A gene chip comprising a nucleic acid molecule of claim 5.

7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5.

8. A nucleic acid vector comprising a nucleic acid molecule of claim 5.

9. A host cell containing the vector of claim 8.

10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.

13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.

14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.

15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.

16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.

17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.

18. A method for treating a disease or condition mediated by a human transporter protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16.

19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.

20. An isolated human transporter peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4.

21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4.

22. An isolated nucleic acid molecule encoding a human transporter peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5.

23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5.